Controllable self-annealing microgel particles for biomedical applications

ABSTRACT

A microporous gel system for certain applications, including biomedical applications, includes an aqueous solution containing plurality of microgel particles including a biodegradable crosslinker. In some aspects, the microgel particles act as gel building blocks that anneal to one another to form a covalently-stabilized scaffold of microgel particles having interstitial spaces therein. In certain aspects, annealing of the microgel particles occurs after exposure to an annealing agent that is endogenously present or exogenously added. In some embodiments, annealing of the microgel particles requires the presence of an initiator such as exposure to light. In particular embodiments, the chemical and physical properties of the gel building blocks can be controlled to allow downstream control of the resulting assembled scaffold. In one or more embodiments, cells are able to quickly infiltrate the interstitial spaces of the assembled scaffold.

RELATED APPLICATIONS

This Application claims priority to U.S. Provisional Patent ApplicationNos. 62/025,844 filed on Jul. 17, 2014, 62/059,463 filed on Oct. 3,2014, and 62/103,002 filed on Jan. 13, 2015. Priority is claimedpursuant to 35 U.S.C. § 119. The above-noted Patent Applications areincorporated by reference as if set forth fully herein.

TECHNICAL FIELD

The technical field relates generally to the field of wound treatment,and in particular, the use of microgel particles and scaffolds includingthe particles for treating and sealing wounds and for tissue fillerapplications.

BACKGROUND

A central concept tied to the generation and regeneration of tissue iscollective cell migration, a process by which entire networks of cellsmove together into an area of development to facilitate the formation offunctional tissue. Researchers have sought to develop would healingagents; however, these materials display batch-to-batch variability andexhibit degradation rates that limit extended structural support forgrowing tissues. Synthetic materials are more tunable than naturalmaterials and their mechanical properties have been engineered to allowuse with a wide range of tissue types. Despite this tunability, however,synthetic injectable biomaterials have been limited to non-porous ornanoporous scaffolds that require physical degradation for cellularmigration through the material. Porous synthetic hydrogels that containpre-formed microscale interconnected pores allow greater cell mobilitywithout the need for degradation, circumventing the trade-off betweencell mobility and material stability inherent to non-porous scaffolds.The typical mode of pore formation includes the toxic removal ofporogens, or the degradation of encapsulated microparticles, whichrequires these constructs to be either cast ex vivo, preventing themfrom seamlessly integrating with the surrounding tissue like aninjectable biomaterial or requires long-term in vivo development toresolve the porous structure. For example, Healionics Corporation hasdeveloped a technology self-described as Sphere Templated AnigiogenicRegeneration (STAR) in which STAR scaffolds are formed by sinteringtogether an array of packed beads of controlled size, casting a polymerinto the interstitial space between the beads, and dissolving away thebeads to yield a pore network of interconnected spherical voids. Asnoted above, however, these conventional processes require the toxicremoval of porogens.

SUMMARY

Human skin wounds are an ever-increasing threat to public health and theeconomy and are very difficult to treat. Physicians, when treating skinwounds, seek to keep the area moist because dry wounds heal much moreslowly than wet ones. To accomplish this, physicians often use ointmentsto fill in the wound, much like filling a pothole with new asphalt.However, these and other conventional approaches to wound healing failto provide an optimal scaffold to allow new tissue to grow. As a result,new tissue growth, if any, is relatively slow and fragile leading tolonger healing times, to the extent timely healing is even possible.

In the context of engineered tissue healing, the instant inventors haveidentified the gold standard of the development of interconnectedmicroporous scaffolds that allow for interconnected cell networks andcollective migration without the need for scaffold degradation orinvasive procedures for implantation is essential for bulk integrationwith the surrounding tissue. In fact, to be most effective, the instantinventors have identified that these materials should facilitatecollective cell migration that mediates regeneration while providingmolecular cues to promote wound healing and niche recognition. Further,the instant inventors have also identified that these materials must beable to be seamlessly replaced by migrating cells and natural matrix,provide a stable structural support prior to replacement, and be easilydelivered and conform to the site of injury to minimize fibrotic andinflammatory responses.

Provided herein are systems, compositions, methods, and devices thatimplement these principles and provide a biomaterial that promotes rapidregeneration of tissue while maintaining structural support ofsurrounding tissue of a wound. Indeed, the present inventors haveachieved solutions to long-felt and unmet medical needs in the field oftissue engineering using a flowable or injectable microgel-based,tailor-made material chemistry and microfluidic fabrication of uniformspherical building blocks, including for example building blocks thewidth of a human hair.

The technology described herein utilizes chemistry to generate tinymicrogels that can be assembled into a large unit, leaving behind a pathfor cellular infiltration. The result is a packed cluster of microscopicsynthetic polymer bodies (e.g., spheres) attached at their surfaces,akin to a jar of gumballs that are stuck together. The cluster creates ascaffold of microporous annealed particles (e.g., a porous gel scaffold)that fills in the wound. New tissue quickly grows into the voids betweenthe microgel particles, and as the microgel particles degrade into thebody, a matrix of newly grown tissue is left where the wound once was.New tissue continues growing until the wound is completely healed.

The microgel systems described herein represents a substantialimprovement over conventional products. For example, the technologiesdescribed herein do not require added growth factors to attract cellsinto the material. The geometry of the described microgel networksentice cells to migrate into the microgel.

The present inventors have demonstrated that the described microgels canpromote the growth of new cells and formation of networks of connectedcells at previously unseen rates. For example, during in vivo studies,significant tissue regeneration was observed in the first 48 hours, withmuch more healing over five days compared to conventional materials inuse today.

The technologies described herein are useful for a wide array ofapplications. For example, the disclosed microgel technology can be usedfor wound applications, including acute damage, like lacerations andsurgical wound closures, and also more chronic applications likediabetic ulcers and large-area burn wounds. The hydrogel scaffoldsdescribed herein can also be useful in trauma situations, such asbattlefields or emergency rooms.

Described herein, in certain aspects, are systems, compositions,methods, and devices comprising a microporous gel that comprises anaqueous solution comprising a plurality of microgel particles and acrosslinker, including for example a biodegradable crosslinker.Microporous gels described herein are flowable and/or injectable and canbe applied in multiple different ways, including for example topicallyor by injection. Injected and/or flowable microporous gels can beinserted transdermally or into deep tissue. Flowable microporous gelscan also be administered topically to the dermis and other tissues.

In one aspect, when an annealing agent is applied to the plurality ofmicrogel particles, the microgel particles form a covalently-stabilizedscaffold of microgel particles having interstitial spaces therein. Incertain applications, the systems, compositions, methods, and devicesare specifically engineered for biomedical applications. In someembodiments, the microporous gel particles further comprise acrosslinker, wherein the crosslinker includes a matrix metalloprotease(MMP)-degradable crosslinker. In one or more embodiments, an annealingagent comprises Factor XIIIa. In further or additional embodiments, theannealing agent comprises Eosin Y, a free radical transfer agent, or acombination thereof.

In some embodiments, the microgel systems, compositions, methods, anddevices further comprises a source of light configured to illuminate amixture of the plurality of microgel particles and the annealing agent.In one or more embodiments, the microporous gel particles comprise celladhesive peptides exposed on a surface thereof. In some embodiments, themicroporous gel particles comprise a K-peptide. In further or additionalembodiments, the microporous gel particles comprise a K-peptide thatcomprises a Factor XIIIa-recognized lysine group. In some embodiments,the microporous gel particles comprise a Q-peptide. In some embodiments,the Q-peptide comprises a Factor XIIIa-recognized glutamine group. Incertain embodiments, the microporous gel particles comprise acrosslinker that is degradable. In certain embodiments, the microporousgel particles comprise interstitial spaces that comprise border surfacesexhibiting negative concavity. In one or more embodiments, thecovalently-stabilized scaffold of microgel particles has a void volumeof from about 10% to about 50%.

In one embodiment, a microporous gel system for biomedical applicationsincludes an aqueous solution containing a plurality of microgelparticles formed with a biodegradable crosslinker such as a matrixmetalloprotease (MMP)-degradable crosslinker and an annealing agent thatwhen applied to the plurality of microgel particles causes the microgelparticles to form a covalently-stabilized scaffold of microgel particleshaving interstitial spaces therein.

In another embodiment, a microporous gel system includes a deliverydevice and a collection of biodegradable microgel particles contained inan aqueous solution and stored in the delivery device. An annealingagent or annealing agent precursor is also stored in the deliverydevice. The delivery device may contain a single or multiplecompartments, depending on the particular embodiment employed.

In another embodiment, a method of treating tissue includes deliveringto the tissue an aqueous-based solution containing a plurality ofmicrogel particles decorated with cell adhesive peptides, wherein themicrogel particles are formed with a biodegradable crosslinker such asmatrix metalloprotease (MMP)-degradable crosslinker. The plurality ofmicrogel particles are exposed to an annealing agent that anneals themicrogel particles to form a covalently-stabilized scaffold of microgelparticles having interstitial spaces therein.

In another embodiment, a microporous gel system for biomedicalapplications includes a collection of microgel particles formed by areaction of a backbone polymer having one or more cell attachmentmoieties, one or more annealing components, and a biodegradable networkcrosslinker component. The microporous gel system includes an endogenousor exogenous annealing agent that links the microgel particles togetherin situ via the annealing components to form a covalently-stabilizedscaffold of microgel particles having interstitial spaces therein.

In another aspect, described herein are systems, compositions, methods,and devices that comprise a delivery device or mechanism and microporousgel. In certain embodiments, the delivery device contains an aqueoussolution comprising a plurality of microgel particles and the annealingagent or an annealing agent precursor. In one or more embodiments, thedelivery device comprises a single compartment delivery devicecontaining the aqueous solution comprising a plurality of microgelparticles and the annealing agent. In one or more embodiments, thedelivery device comprises a multiple (e.g., double) compartment deliverydevice, wherein one compartment contains the aqueous solution containingplurality of microgel particles and a first annealing agent precursorand the second compartment contains the aqueous solution containingplurality of microgel particles and a second annealing agent precursor.In certain embodiments, microporous gels further comprise a(MMP)-degradable crosslinker that comprises at least one D-amino acid.In further or additional embodiments, the microgel particles comprise a(MMP)-degradable crosslinker comprises a plurality of D-amino acids.

In yet another aspect, described here is a microporous gel systemcomprising: a delivery device; a plurality biodegradable microgelparticles contained in an aqueous solution and stored in the deliverydevice; and an annealing agent or annealing agent precursor stored inthe delivery device. In one or more embodiments, the microporous gelparticles further comprise a collection of biodegradable microgelparticles of two or more types that are contained in an aqueous solutionand stored in the delivery device. In certain embodiments, the deliverydevice comprises two compartments, biodegradable microgel particles arestored in each of the two compartments, and a first annealing precursoris stored in one compartment and a second annealing precursor is storedin the other compartment, wherein the annealing agent is formed by thepresence of both the first and second annealing precursors. In one ormore embodiments, the delivery device comprises a single compartment andthe collection of biodegradable microgel particles and the annealingagent are both stored in the single compartment. In still further oradditional embodiments, the annealing agent comprises a photoinitiatorand a free radical transfer agent stored in the single compartment. In afurther or additional embodiment, the microporous gel system furthercomprises a light-emitting device configured to illuminate a mixture ofthe collection of biodegradable microgel particles and the annealingagent. In certain embodiments, the microgel particles comprisesubstantially monodisperse spheres. In one or more embodiments, thesubstantially monodisperse spheres have a diameter within the range offrom about 30 micrometers to about 150 micrometers. In further oradditional embodiments, the microgel particles are covalently linked toanother after annealing.

Provided in another aspect is a method of treating tissue comprising:delivering to the tissue an aqueous-based solution containing aplurality of microgel particles; and exposing the plurality of microgelparticles to an annealing agent that anneals the microgel particles toform a covalently-stabilized scaffold of microgel particles havinginterstitial spaces therein. In some embodiments, the plurality ofmicrogel particles is decorated with cell adhesive peptides, and whereinthe microgel particles are formed with a matrix metalloprotease(MMP)-degradable crosslinker. In one or more embodiments, the annealingagent is delivered to the tissue. In some embodiments, the annealingagent is present within the tissue. In yet additional embodiments, themethod further comprises initiating the annealing of the microgelparticles with exposure to light. In some embodiments, the wavelength oflight is in the visible range. In some embodiments, the wavelength oflight is in the infrared range. In one or more embodiments, theaqueous-based solution and the annealing agent are deliveredsimultaneously. In some embodiments, the aqueous-based solution and theannealing agent are delivered sequentially. In still further oradditional embodiments, the microgel particles comprise atherapeutically active chemical compound. In certain embodiments, themicrogel particles expose or elute the chemical compound to the tissue.In one or more embodiments, the tissue comprises a site of cosmeticreconstruction, chronic wound development, acute tissue damage, or atissue gap caused by surgical incision. In yet additional embodiments,the (MMP)-degradable crosslinker comprises D-amino acid.

In another aspect, provided is a microporous gel system or devicecomprising: a collection of microgel particles comprising a backbonepolymer having one or more cell attachment moieties, one or moreannealing components, and one or more biodegradable network crosslinkercomponents; and an endogenous or exogenous annealing agent that linksthe microgel particles together in situ via the annealing components toform a covalently-stabilized scaffold of microgel particles havinginterstitial spaces therein. In certain embodiments, the backbonepolymer comprises poly(ethylene glycol) vinyl sulfone. In one or moreembodiments, the one or more cell attachment moieties comprise a RGDpeptide or a fragment thereof, fibronectin or a fragment thereof,collagen or a fragment thereof, or laminin or a fragment thereof. Insome embodiments, the one or more cell attachment moieties comprise aRGD peptide or a fragment thereof. In an embodiment, the one or morecell attachment moieties comprise SEQ ID NO: 3 or a fragment thereof. Infurther or additional embodiments, the one or more annealing componentscomprise a K-peptide and a Q-peptide. In certain embodiments, theK-peptide comprises a Factor XIIIa-recognized lysine group and theQ-peptide comprises a Factor XIIIa-recognized glutamine group. In someembodiments, the biodegradable network crosslinker component comprises amatrix metalloprotease (MMP)-degradable crosslinker. In one or moreembodiments, the (MMP)-degradable crosslinker comprises D-amino acid. Incertain embodiments, the collection of microgel particles comprisesmicrogel particles of two or more types. In one or more embodiments, themicrogel particles of a first type comprise (MMP)-degradable crosslinkercomprising D-amino acid, and microgel particles of a second typecomprise (MMP)-degradable crosslinker comprising only L-amino acid. Inone or more embodiments, the system or device comprises a singlecompartment delivery device containing the collection of microgelparticles and the annealing agent. In one or more embodiments, thesystem or device further comprises a double compartment delivery device,wherein one compartment contains the aqueous solution containingplurality of microgel particles and a first annealing agent precursorand the second compartment contains the aqueous solution containingplurality of microgel particles and a second annealing agent precursor,wherein the annealing agent is formed by the presence of the first andsecond annealing agent precursors.

In an additional aspect, described is a method of treating tissuecomprising: delivering to the tissue a first layer of microgel particlesdecorated with cell adhesive peptides, wherein the microgel particlesare formed with a biodegradable crosslinker; exposing the first layer toan annealing agent that anneals the microgel particles to form acovalently-stabilized scaffold of microgel particles having interstitialspaces therein; delivering to the tissue a second layer of microgelparticles decorated with cell adhesive peptides, wherein the microgelparticles are formed with a biodegradable crosslinker and wherein themicrogel particles in the second layer differ in one of a physicalproperty or chemical composition as compared to the microgel particlesin the first layer; and exposing the second layer to an annealing agentthat anneals the microgel particles to form a covalently-stabilizedscaffold of microgel particles having interstitial spaces therein. Inone or more embodiments, the microgel particles in the second layer havea different size. In yet additional embodiments, the microgel particlesin the second layer have a different shape. In one or more embodiment,the microgel particles in the second layer have a different stiffness.In certain embodiments, the microgel particles in the second layerhaving a chemical component different from a chemical component in thefirst layer. In further or additional embodiment, the microgel particlesin the second layer having a chemical component of a differentconcentration from the same chemical component in the first layer.

In another aspect, provided is method of treating tissue comprising:delivering to the tissue an aqueous-based solution containing aplurality of microgel particles decorated with cell adhesive peptides,wherein the microgel particles are formed with a biodegradablecrosslinker, exposing the plurality of microgel particles to anannealing agent that anneals the microgel particles to form acovalently-stabilized scaffold of microgel particles having interstitialspaces therein.

In another embodiment, a method of treating tissue includes deliveringto the tissue a first layer of microgel particles decorated with celladhesive peptides, wherein the microgel particles are formed with abiodegradable crosslinker. The first layer is exposed to an annealingagent that anneals the microgel particles to form acovalently-stabilized scaffold of microgel particles having interstitialspaces therein. A second layer of microgel particles decorated with celladhesive peptides is delivered to the tissue, wherein the microgelparticles are formed with a biodegradable crosslinker and wherein themicrogel particles in the second layer differ in one of a physicalproperty or chemical composition as compared to the microgel particlesin the first layer. The second layer is exposed to an annealing agentthat anneals the microgel particles to form a covalently-stabilizedscaffold of microgel particles having interstitial spaces therein.

In another embodiment, a method of treating tissue includes deliveringto the tissue an aqueous-based solution containing a plurality ofmicrogel particles decorated with cell adhesive peptides, wherein themicrogel particles are formed with a biodegradable crosslinker. Theplurality of microgel particles are exposed to an annealing agent thatanneals the microgel particles to form a covalently-stabilized scaffoldof microgel particles having interstitial spaces therein.

In yet an additional aspect, described is a method of making microgelparticles comprising: providing a water-in-oil droplet generatingmicrofluidic device having a plurality of input channels leading to acommon channel and a pair of oil-pinching channels intersecting with thecommon channel at a downstream location flowing a first pre-polymersolution containing a polymer backbone modified with oligopeptides intoa first input channel; flowing a second solution containing abiodegradable crosslinker into a second input channel; flowing an oiland a surfactant into the pair of oil pinching channels to form dropletscontaining the first pre-polymer solution and the second solution; andcollecting microgel particles formed by cross-linking of the droplets.In another embodiment, the method further comprises a third inputchannel interposed between the first input channel and the second inputchannel, wherein a third inert solution containing a pre-polymer isflowed into the third input channel. In one or more embodiments, themethod further comprises sheathing the generated droplets with anadditional pair of sheathing channels located downstream of a locationwhere the pair of oil pinching channels intersect with the commonchannel, wherein the additional pair of sheathing channels carries oiland a surfactant at a higher concentration than the surfactant containedin the upstream pair of oil pinching channels. In one embodiment, themethod further comprises centrifuging the collected microgel particles.In another aspect, the method comprises reducing the free water volumecontent of the centrifuged microgel particles. In one or moreembodiments, the method comprises storing the collected microgelparticles for an extended period of time (e.g., months to years).

In still another embodiment, a method of making microgel particlesincludes providing a water-in-oil droplet generating microfluidic devicehaving a plurality of input channels leading to a common channel and apair of oil-pinching channels intersecting with the common channel at adownstream location. A first pre-polymer solution containing a polymerbackbone modified with oligopeptides is flowed into a first inputchannel. A second solution containing a biodegradable crosslinker isflowed into a second input channel. An oil and a surfactant are flowedinto the pair of oil pinching channels to form droplets containing thefirst pre-polymer solution and the second solution. Microgel particlesare formed by cross-linking of the droplets which are then collected.

Other objects, features and advantages of the present disclosure willbecome apparent to those skilled in the art from the following detaileddescription. It is to be understood, however, that the detaileddescription and specific examples, while indicating some embodiments ofthe present disclosure are given by way of illustration and notlimitation. Many changes and modifications within the scope of thepresent disclosure may be made without departing from the spiritthereof, and the disclosure includes all such modifications. Moreoveraspects of one embodiment may be utilized in other, differentembodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the disclosure are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present disclosure will be obtained by reference tothe following detailed description that sets forth illustrativeembodiments, in which the principles of the disclosure are utilized, andthe accompanying drawings of which:

FIG. 1 illustrates a portion of a scaffold formed from a plurality ofannealed microgel particles.

FIG. 2A illustrates an exemplary method of injecting microgel particlesinto a wound site for healing the same.

FIG. 2B schematically illustrates an exemplary annealing reactionbetween different microgel particles potentiated by linkers on thesurface of the microgel particles.

FIG. 2C illustrates an exemplary process of tissue infiltration into ascaffold formed within a delivery site on tissue, where the boundarybetween the tissue and the microgels represents any interface betweenthem, where cells can pass through the interface moving inwards from thetissue or outward toward the tissue from the microgels.

FIG. 3A illustrates a top down view of a microfluidic device accordingto one embodiment used to generate a plurality of microgel particles aspart of a microporous gel system.

FIG. 3B illustrates a magnified view of the droplet generation regionand downstream oil/surfactant pinching region (see box region in FIG.3A).

FIG. 3C illustrates magnified, perspective views of two branch channelsillustrated in FIG. 3A.

FIG. 3D illustrates a side view of the microfluidic device of FIG. 3Aaccording to one embodiment.

FIG. 3E illustrates a photograph taken of a reduction to practice of thescheme illustrated in FIG. 3B where fluorescent solution on the leftcontains crosslinker, the fluorescent solution on the right containspolymer and reaction buffer, and the middle stream contains an inertliquid solution to prevent mixing of left and right solutions prior todroplet segmentation. Bright fluorescence between middle and rightstreams illustrates pH change in the middle stream due to diffusion ofreaction buffer.

FIG. 3F illustrates a photograph of a reduction to practice of thescheme illustrated in FIG. 3B and FIG. 3E, while also showing the lightmicroscopic view of droplet segmentation after the pinching oil streamsare introduced.

FIG. 4A illustrates a top down view of a microfluidic device accordingto another embodiment used to generate a plurality of microgel particlesas part of a microporous gel system.

FIG. 4B illustrates that in the droplet segmentation region, mineral oilwith 0.25% Span® 80 pinches and segments PEG pre-gel, and downstream a5% Span® 80 solution in mineral oil mixes and prevents downstreamcoalescence of microgels before complete gelation.

FIG. 4C illustrates droplets do not recombine during incubation in thebifurcation region and exit from the microchannel to the collectionwell.

FIG. 5 illustrates an exemplary microfluidic T-junction that may be usedto generate microgel droplets according to one embodiment.

FIG. 6A illustrates an exemplary dispensing device in the form of adouble-barreled syringe according to one embodiment.

FIG. 6B illustrates an exemplary dispensing device in the form of asingle-barreled syringe according to another embodiment.

FIG. 6C illustrates an exemplary dispensing device in the form of a tubethat holds the microgel particles according to one embodiment.

FIG. 7A illustrates hematoxylin and eosin staining (H&E staining) oftissue sections in SKH1-Hr^(hr) mice for tissue injected with thescaffold (Microporous Annealed Particle or “MAP” scaffold) as well asthe non-porous control twenty-four (24) hours after injection.

FIG. 7B illustrates a graph of wound closure (%) as a function of dayspost-injection. This graphs shows that over a five (5) day period thereis statistically significant improvement in the wound closure rates forusing the scaffolds when compared to non-porous bilateral controls(N=5).

FIG. 7C illustrate representative images of wound closure during a 5-dayin vivo wound healing model in SKH1-Hr^(hr) mice comparing the gelscaffold (left panels) to a non-porous PEG gel control (right panels).

FIG. 7D illustrates representative images of wound closure during 7-dayin vivo BALB/c experiments. After 7 days in vivo, the scaffolds promotesignificantly faster wound healing than the no treatment control, thegels lacking the K and Q peptides, the non-porous PEG gel, and fasterwound healing than the precast porous gel. Porous gels created ex vivoto precisely match the wound shape using the canonical, porogen-based,casting method showed appreciable wound healing rates, comparable to thescaffolds, but lacking injectability (N≧5).

FIG. 7E is a bar graph illustrating wound closure quantification datafrom BALB/c in vivo wound healing for each treatment categorycorresponding to FIG. 7D. All data are presented as average+/−SEM.Statistical significance performed using standard two-tailed t-test (*:p<0.05; **p<0.01).

FIG. 7F illustrates traces of wound bed closure during 7 days in vivofor each treatment category corresponding to FIG. 7D and FIG. 7E.

FIG. 7G illustrates how the microgel particle-containing solution orslurry can be injected using a syringe device (e.g., 25 Gauge syringe)like that of FIG. 6A or 6B into a treatment site where the microgelconforms to the shape of the injection site (e.g., in this case astar-shaped laser cut acrylic mold) and subsequent annealing of thescaffold into the star shape.

FIGS. 8A and 8B illustrate stained microscopic images of damaged tissue(i.e., wound site) that has been treated with the microgel scaffold(FIG. 8A) and with no treatment or “sham” (FIG. 8B) in a mouse modeltwenty-one (21) days after skin excision and gel application. The scarreduction enabled by the microgel scaffold can clearly be seen in FIG.8A. Squares indicate hair follicles and oil glands (sebaceous glands) inthe reforming tissue after gel application to a wound. Circles indicateremaining microgel particles in the reforming tissue.

FIG. 8C illustrates a graph showing the epidermal thickness for thetissue treated with the sham as well as tissue treated with the gelscaffold.

FIG. 8D illustrates a graph showing the number of sebaceous glands forthe tissue treated with the sham as well as tissue treated with the gelscaffold.

FIG. 8E illustrates a graph showing the number of hair follicles for thetissue treated with the sham as well as tissue treated with the gelscaffold.

FIG. 8F illustrates a graph showing the scar width for the tissuetreated with the sham as well as tissue treated with the gel scaffold.

FIG. 8G illustrates a graph showing the number of milial cysts for thetissue treated with the sham as well as tissue treated with the gelscaffold.

FIG. 9A illustrates a graph of storage modulus as a function of timepost-mixing for different gelation kinetics (pH and temperature). pH8.25 at 25 degrees Celsius is represented by the bottom line in thegraph; pH 8.8 at 25 degrees Celsius is represented by the top line inthe graph; and pH 8.25 at 37 degrees Celsius is represented by themiddle line in the graph.

FIG. 9B illustrates different hydrogel weight percentages were used toproduce different stiffness materials on the x-axis. The graphillustrated Storage Modulus (Pa) for various hydrogel weightpercentages.

FIG. 9C illustrates different crosslinker stoichiometries that were usedto produce different stiffness values in the resultant gel on thex-axis. The graph illustrated Storage Modulus (Pa) as a function of ther-ratio of free crosslinker ends (−SH) to vinyl groups (−VS) on the PEGmolecule.

FIG. 9D illustrates a graph of the % degradation as a function of timefor both the non-porous control (bottom line of the graph) as well as aporous gel described herein (top line of the graph).

FIG. 9E illustrates SEM images of a scaffold annealed with FXIIIa at 200μm (top panel) or 100 μm (bottom panel).

FIG. 9F illustrates SEM images of microgel particles without FXIIIa at200 μm (top panel) or 100 μm (bottom panel). Un-annealed microgelparticles are seen in FIG. 9F.

FIG. 10 shows a microgel fabricated using the described technique, wherethe surface of the microgel has been augmented with a fluorescent bovineserum albumin (BSA) protein (outer perimeter) through the use ofphosphine-azide ‘click’ chemistry. Further, nanoparticles (500 nm) areembedded within the microgel during microfluidic fabrication.

FIG. 11 illustrates an exemplary method of treating damaged tissue usingthe microporous gel system described herein. Microgel particles areapplied (top panel), optionally, an applicator is utilized (secondpanel), annealing of microgel particles is initiated to form a scaffold(third panel) and improved wound healing is observed (bottom panel).

FIG. 12A illustrates fluorescent images demonstrating the formation of3D cellular networks during six days of culture in porous gel scaffoldsin vitro as well as non-porous gels after 6 days. (350 Pa: bulk modulusidentical to porous gel scaffolds, 600 Pa: microscale modulus matched toindividual microgels).

FIG. 12B illustrates a graph of cell survival twenty-four (24) hourspost annealing is greater than 93% across three cell lines representingdifferent human tissue types. HDF: Human dermal fibroblasts, AhMSC:Adipose-derived human mesenchymal stem cells, BMhMSC: Bonemarrow-derived human mesenchymal stem cells.

FIG. 13A illustrates an exemplary method for combining living cells withpreformed microgel particles prior to annealing. The microgel particlesare annealed to one another, entrapping the living cells within theinterconnected microporous network created upon microgel annealing.

FIGS. 13B-D are photographic images illustrating that microgel particlesolutions combined with living cells are moldable to macro-scale shapes,and can be injected to form complex shapes that are maintained afterannealing. FIG. 13B illustrates an exemplary in vitro syringe injection.FIG. 13C illustrates an exemplary in vitro shape molding. FIG. 13Dillustrates an exemplary in vitro annealed scaffold. FIG. 13Eillustrates microgel particles are moldable to macro-scale shapes andcan be performed in the presence of live cells (indicated by arrowspointing to fluorescent HEK-293T cells).

FIG. 14A illustrates a graph showing that varying sizes of microgelparticles can be synthesized over a range of frequencies of productionin an exemplary embodiment.

FIG. 14B illustrates that providing a high inlet pressure to eachsolution inlet (where the oil inlets are exceeding 30 Psi) enables anincrease in production frequency in another exemplary embodiment.

FIG. 14C illustrates a graph showing high precision fabrication ofmicrogel building blocks allows creation of defined gel scaffolds.Different building block sizes allow for deterministic control overresultant micro-porous network characteristics, presented here as medianpore sizes+/−standard deviation (SD).

DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

In the description of the preferred embodiment, reference is made to theaccompanying drawings which form a part hereof, and in which is shown byway of illustration a specific embodiment in which the subject matterdescribed herein may be practiced. It is to be understood that otherembodiments may be utilized and structural changes may be made withoutdeparting from the scope and spirit of the inventive subject matterdescribed herein. Further, various aspects of different embodiments maybe utilized with other embodiments described herein without departingfrom the scope of the invention.

In one aspect of the subject matter described herein, a solid microgelscaffold for biomedical applications such as wound healing is disclosedthat is formed when a plurality of microgel particles are annealed toone another in an annealing reaction. The annealing reaction, in oneaspect of the subject matter described herein forms covalent bondsbetween adjacent microgel particles. For example, in the post-annealedstate, the scaffold forms a three-dimensional structure that conforms tothe site of application or delivery. Because of the imperfect packing ofthe microgel particles, the annealed scaffold formed from the particlesincludes interstitial spaces formed therein where cells can migrate,bind, and grow. The formed scaffold structure is porous upon annealingin the wound or other delivery site (unlike the non-porous solidscaffold provided by fibrin-based products). This porosity includes theinterstitial spaces mentioned above as well as nanoscopic pores that maybe created or formed in the particles themselves. The micro-porosity ofthe scaffold structure allows for high diffusivity of nutrients, cellgrowth and differentiation factors, as well as cell migration, ingrowth,and penetration. The microporosity of the scaffold provides foraccelerated healing or improved therapeutic delivery of drugs ormedicaments over conventional fibrin glue, hyper-branched polymers, orpolymers with degradable crosslinker options, because of the enhancedcell migration through interstitial spaces while maintaining overallscaffold integrity. In addition, by not limiting the biomaterial tonatural materials, the degradation profile and physical properties(e.g., stiffness, internal diffusivity, etc.) are improved, for example,by having a larger available range and a wider array of biologicalsignals or therapeutically-active chemicals can be included within thematerial (e.g., antibiotics, steroids, growth factors, and the like canbe loaded into the scaffold). Furthermore, the release or elution of thedrugs, compounds, or other material to trigger or control biologicalactivity, in certain embodiments, can be tuned through modification ofthe desired biomaterial. The signal compounds or molecules discussedabove may be exposed to the tissue during the healing process or upondegradation of the scaffold. The signal compounds or molecules may alsobe released or eluted into the affected area after initial placement ofthe scaffold at the delivery site.

One advantage of the subject matter described herein beyond methods suchas the STAR™ technology is that the formation of a scaffold occurs invivo, allowing it to completely fill the desired space and be tuned tobind (chemically or otherwise) to the surrounding tissue. In addition,the pre-delivery formation of the microgel particles allows forcontrolled mechanical tunability of the resultant formed scaffold tomatch the properties of the surrounding tissue. These capabilitiesresult in a better seal and overall integration with the tissue. Greaterintegration results in decreased possibility of material failure andenhanced long-term regeneration. This also helps prevent contaminationfrom the environment. Moreover, the microporous nature of the annealedscaffold is beneficial to reduce immune foreign body response to thescaffold.

FIG. 1 illustrates a portion of the formed three dimensional scaffold 10that is formed by a plurality of annealed microgel particles 12. Thescaffold 10 includes interstitial spaces therein 14 that are voids thatform micropores within the larger scaffold 10. The interstitial spaces14 have dimensions and geometrical profiles that permit theinfiltration, binding, and growth of cells. It should be appreciatedthat the microporous nature of the scaffold 10 disclosed herein involvesa network of interstitial spaces or voids 14 located between annealedmicrogel particles 12 that form the larger scaffold structure. In oneembodiment, the interstitial spaces or voids 14 created within thescaffold 10 exhibit negative concavity (e.g., the interior void surfaceis convex). FIG. 1 illustrates an exemplary void 14 with void walls 16exhibiting negative concavity. The negative concavity is caused becausethe microgel particles 12 that are annealed to one another are generallyor substantially spherical in shape in one preferred embodiment. Thisallows for the packing of microgel particles 12 that, according to oneembodiment, produces a low void volume fraction between about 10% andabout 50% and, in another embodiment between about 26% to about 36%.While the void volume fraction is low, the negative concavity exhibitedin certain embodiments within the network of voids 14 provides arelatively high surface area to void volume for cells to interact with.For a given volume of cells, they would then, on average, be exposed toeven more and larger surfaces (e.g., on the void walls 16) to interactwithin the network of voids in the scaffold 10.

It is important to note that the void network consists of regions wheremicrogel surfaces are in close proximity (e.g., near neighboringannealed microgel particles 12) leading to high surface area adhesiveregions for cells to adhere and rapidly migrate through, whileneighboring regions further in the gaps between microgel particles 12have a larger void space that can enable cell and tissue growth in thisspace. Therefore the combined adjacency of the tight void areas and morespacious void gaps is expected to have a beneficial effect on tissueingrowth and regrowth, compared to either entirely small voids or alllarger voids.

Note that in the embodiment described above, the negative concavityresults due to the spherical shape of the microgel particles 12. Inother embodiments, the microgel particles 12 might not be spherical inshape. Other non-spherical shapes may still be used in the scaffold 10.Still referring to FIG. 1, the scaffold 10 is formed by microgelparticles 12 that are secured to one another via annealing surfaces 17.As explained herein, the annealing surfaces 17 are formed either duringor after application of the microgel particles 12 to the intendeddelivery site.

The scaffold 10 may be used for various applications, including avariety of medical applications such as military field medicine, medicaltrauma treatment, post-surgical closure, burn injuries, inflammatory andhereditary and autoimmune blistering disorders, etc. In one or moreembodiments, the scaffold 10 is used as a tissue sealant (e.g., an acutewound-healing substance, surgical sealant, topical agent for partialthickness, full thickness, or tunneling wounds, pressure ulcers, venousulcers, diabetic ulcers, chronic vascular ulcers, donor skin graftsites, post-Moh's surgery, post-laser surgery, podiatric wounds, wounddehiscence, abrasions, lacerations, second or third degree burns,radiation injury, skin tears, and draining wounds, and the like). FIGS.2A-2C illustrate an embodiment, where the scaffold 10 is used to treat awound site 100 formed in tissue 102 of a mammal. In certain embodiments,the scaffold 10 is used for immediate treatment of acute wounds. Inacute wounds, the scaffold 10 provides several benefits, including arapid method to seal wounds 100, prevent trans-epidermal water loss,provide cells or medication(s), and enhance the healing of skin wounds(e.g., surgical sites, burn wounds, ulcers) to provide more naturaltissue development (e.g., avoiding the formation of scar tissue). Oneparticular benefit of the scaffold 10 is the ability of the scaffold 10to reduce or minimize the formation of scar tissue. The scaffold 10provides a more effective alternative to tissue glues and other currentinjectable tissue fillers and adhesives.

As seen in FIG. 2A, microgel particles 12 are delivered to the woundsite 100 followed by the initiation of the annealing reaction to annealthe microgel particles 12 to one another to form the scaffold 10. Asseen in FIG. 2A, the wound site 100 is sealed by the scaffold 10 and astime progresses, the wound site 100 is healed into normal tissue (seealso FIG. 11). FIG. 2B illustrates how adjacent microgel particles 12(particle A and particle B) undergo chemical or enzymatic initiation ofthe annealing reaction to form an annealing surface 17 between microgelparticles 12. FIG. 2C illustrates a magnified view illustrating how thescaffold 10 acts as a structural support yet permits the tissueinfiltration and biomaterial resorption due to the porous nature of thescaffold 10. A cell 106 is illustrated infiltrating the interstitialspaces formed within the scaffold 10.

The scaffold 10 may also be used in a regenerative capacity, forexample, applied to tissue for burns, acute and chronic wounds, and thelike. In one embodiment, the scaffold 10 is used for chronic wounds. Inchronic wounds, where the normal healing process is inhibited, thescaffold 10 can be used not only to seal wounds, but also to removeexcess moisture, and apply medication(s), including cellular therapiesthat can assist in promoting the normal wound healing process. In thecase of tissue filler applications for volume loss related to aging,lipoatrophy, lipodystrophy, dermal scarring, or superficial or deeprhytides, injection of the microgel particles 12 directly into thedermis via needle or cannula may be used to improve tissue contour,tissue loss, or tissue displacement. Because cells used in regenerativemedicine can grow within the microgel particles 12, cells (e.g.,mesenchymal stem cells, fibroblasts, etc.) may be included as a therapyby initially polymerizing the cells (1-20 cells) within microgelparticles, or cells may be initially adhered to microgel particles, orcells may be introduced with the microgel particle solution(non-adhered), prior to annealing in situ in tissue.

The scaffold 10 may also be used for in vitro tissue growth,three-dimensional (3D) matrices for biological science studies, andcosmetic and dermatologic applications. For example, cancer cells couldbe seeded along with the microgel precursors and once annealed couldallow for rapid 3D growth of tumor spheroids for morephysiologically-relevant drug testing without the need for matrixdegradation as would be required for other 3D culture gels (e.g.,Matrigel®). It is expected that the rapid ability to form contactsbetween cells in the 3D matrix of the annealed gel will enhance growthand formation of micro-tissues from a single cell type or multiple celltypes which can be used to screen for drugs or test cosmetics. Epidermallayers can form over the surface of a scaffold 10, which could allowtesting of drugs or cosmetics on a more skin-like substitute compared toanimal models. Previous 3D culture materials either can enable cellseeding within the gel uniformly through the volume, but not maintaincell-cell contacts because of the lack of porosity, or create porositybut require cells to be seeded following fabrication and migrate intothe scaffold.

As explained herein, while the annealed scaffold 10 generally forms adefined structure, the precursor materials prior to final annealing isflowable and can be delivered as paste, slurry, or even injected to thedelivery site of interest. Other injectable hydrogels can provide ascaffold for in situ tissue regrowth and regeneration, however theseinjected materials require gel degradation prior to tissue reformationlimiting their ability to provide physical support. The injectablemicroporous gel system described herein circumvents this challenge byproviding an interconnected microporous network for simultaneous tissuereformation and material degradation.

Microfluidic formation enables substantially monodisperse microgelparticles 12 to form into an interconnected microporous annealedparticle scaffold 10 (in one aspect of the subject matter describedherein), thereby enabling the controlled chemical, physical, andgeometric properties of the microgel particles 12 (e.g., buildingblocks), to provide downstream control of the physical and chemicalproperties of the assembled scaffold 10. In vitro, cells incorporatedduring scaffold 10 formation proliferate and form extensivethree-dimensional networks within forty-eight (48) hours. In vivo, theinjectable gel system that forms the scaffold 10 facilitates cellmigration resulting in rapid cutaneous tissue regeneration and tissuestructure formation within five (5) days. The combination ofmicroporosity and injectability achieved with the scaffolds 10 enablesnovel routes to tissue regeneration in vivo and tissue creation de novo.

FIG. 2A illustrates the scaffold 10 formed within a wound site 100.Successful materials for tissue regeneration benefit from preciselymatching the rate of material degradation to tissue development. Ifdegradation occurs too quickly then insufficient scaffolding will remainto support tissue ingrowth. Conversely, a rate that is too slow willprevent proper tissue development and can promote fibrosis and/or immunerejection. Tuning of degradation rates based on local environment hasbeen approached using hydrolytically and enzymatically degradablematerials. However, decoupling loss of material mechanical stabilitywith cellular infiltration has proven extremely challenging. Promotionof cellular infiltration into the material can also be approached usinga lightly crosslinked matrix, however this often results in mechanicalmismatch with surrounding tissues and poor material stability.Alternatively, the hydrogel degradation rate can be tuned by alteringthe polymeric backbone identity or crosslinking density, matching therates of degradation and tissue formation. Although these techniques canbe tuned to address specific applications of injectable hydrogels, theydo not provide a robust pathway to achieve bulk tissue integration thatdoes not rely on loss of material stability.

Every wound site is unique in its physical, chemical, and degradationrequirements for functional tissue regeneration, requiring a materialstrategy that is robust to a variety of challenging environments. Themicroporous gel system and the resulting scaffold 10 that is created asdescribed herein circumvents the need for material degradation prior totissue ingrowth by providing a stably linked interconnected network ofmicropores for cell migration and bulk integration with surroundingtissue. The microporous gel system achieves these favorable features by,according to one embodiment, using the self-assembly of microgelparticles 12 as “building blocks” or “sub-units” formed by microfluidicwater-in-oil droplet segmentation. According to one embodiment, themicrogel particles 12 formed in this manner are substantiallymonodisperse. The microgel particles 12 can be injected and molded intoany desired shape. Lattices of microgel particles 12 are then annealedto one another via surface functionalities to form an interconnectedmicroporous scaffold 10 either with or without cells present in theinterconnected porous networks. The scaffold 10 preferably, in oneembodiment, includes covalently linked microgel particles 12 that form athree-dimensional scaffolding 10 for tissue regeneration and ingrowth.

By combining injectability and microporosity, the microporous gel systemprovides an ideal biomaterial scaffold for efficient cellular networkformation in vitro and bulk tissue integration in vivo. The modularmicroporous gel system also provides mechanical support for rapid cellmigration, molecular cues to direct cell adhesion, and resorption duringand after tissue regeneration. Through microfluidic fabrication, thechemical, physical, and geometric properties of the microgel particles12 can be predictably and uniformly tailored, allowing for downstreamcontrol of the properties of the emergent scaffolds 10. The novelbuilding block-based approach in which robustly achieved imperfectself-assembly is desirable to achieve microporosity fundamentallychanges the use and implementation of hydrogels as tissue mimeticconstructs, providing a philosophical change in the approach toinjectable scaffolding for bulk tissue integration.

In one aspect of the subject matter described herein, the microporousgel system uses microgel particles 12 having diameter dimensions withinthe range from about 5 μm to about 1,000 μm. The microgel particles 12may be made from a hydrophilic polymer, amphiphilic polymer, syntheticor natural polymer (e.g., poly(ethylene glycol) (PEG), poly(propyleneglycol), poly(hydroxyethylmethacrylate), hyaluronic acid (HA), gelatin,fibrin, chitosan, heparin, heparan, and synthetic versions of HA,gelatin, fibrin, chitosan, heparin, or heparan). In one embodiment, themicrogel particle 12 is made from any natural (e.g., modified HA) orsynthetic polymer (e.g., PEG) capable of forming a hydrogel. In one ormore embodiments, a polymeric network and/or any other support networkcapable of forming a solid hydrogel construct may be used. Suitablesupport materials for most tissue engineering/regenerative medicineapplications are generally biocompatible and preferably biodegradable.Examples of suitable biocompatible and biodegradable supports include:natural polymeric carbohydrates and their synthetically modified,crosslinked, or substituted derivatives, such as gelatin, agar, agarose,crosslinked alginic acid, chitin, substituted and cross-linked guargums, cellulose esters, especially with nitrous acids and carboxylicacids, mixed cellulose esters, and cellulose ethers; natural polymerscontaining nitrogen, such as proteins and derivatives, includingcross-linked or modified gelatins, and keratins; vinyl polymers such aspoly(ethyleneglycol)acrylate/methacrylate/vinylsulfone/maleimide/norbornene/allyl, polyacrylamides, polymethacrylates,copolymers and terpolymers of the above polycondensates, such aspolyesters, polyamides, and other polymers, such as polyurethanes; andmixtures or copolymers of the above classes, such as graft copolymersobtained by initializing polymerization of synthetic polymers on apreexisting natural polymer. A variety of biocompatible andbiodegradable polymers are available for use in therapeuticapplications; examples include: polycaprolactone, polyglycolide,polylactide, poly(lactic-co-glycolic acid) (PLGA), andpoly-3-hydroxybutyrate. Methods for making networks from such materialsare well-known.

In one or more embodiments, the microgel particles 12 further includecovalently attached chemicals or molecules that act as signalingmodifications that are formed during microgel particle 12 formation.Signaling modifications includes the addition of, for example, adhesivepeptides, extracellular matrix (ECM) proteins, and the like. Functionalgroups and/or linkers can also be added to the microgel particles 12following their formation through either covalent methods ornon-covalent interactions (e.g., electrostatic charge-chargeinteractions or diffusion limited sequestration). Crosslinkers areselected depending on the desired degradation characteristic. Forexample, crosslinkers for the microgel particles 12 may be degradedhydrolytically, enzymatically, photolytically, or the like. In oneparticular preferred embodiment, the crosslinker is a matrixmetalloprotease (MMP)-degradable crosslinker.

Examples of these crosslinkers are synthetically manufactured ornaturally isolated peptides with sequences corresponding to MMP-1 targetsubstrate, MMP-2 target substrate, MMP-9 target substrate, randomsequences, Omi target sequences, Heat-Shock Protein target sequences,and any of these listed sequences with all or some amino acids being Dchirality or L chirality. In another embodiment, the crosslinkersequences are hydrolytically degradable natural and synthetic polymersconsisting of the same backbones listed above (e.g., heparin, alginate,poly(ethyleneglycol), polyacrylamides, polymethacrylates, copolymers andterpolymers of the listed polycondensates, such as polyesters,polyamides, and other polymers, such as polyurethanes).

In another embodiment, the crosslinkers are synthetically manufacturedor naturally isolated DNA oligos with sequences corresponding to:restriction enzyme recognition sequences, CpG motifs, Zinc fingermotifs, CRISPR or Cas-9 sequences, Talon recognition sequences, andtranscription factor-binding domains. Any of the crosslinkers from thelisted embodiments one are activated on each end by a reactive group,defined as a chemical group allowing the crosslinker to participate inthe crosslinking reaction to form a polymer network or gel, where thesefunctionalities can include: cysteine amino acids, synthetic andnaturally occurring thiol-containing molecules, carbene-containinggroups, activated esters, acrylates, norborenes, primary amines,hydrazides, phosphenes, azides, epoxy-containing groups, SANPAHcontaining groups, and diazirine containing groups.

In one embodiment, the chemistry used to generate microgel particles 12allows for subsequent annealing and scaffold 10 formation throughradically-initiated polymerization. This includes chemical-initiatorssuch as ammonium persulfate combined with Tetramethylethylenediamine.Alternatively, photoinitators such as Irgacure® 2959 or Eosin Y togetherwith a free radical transfer agent such as a free thiol group (used at aconcentration within the range of 10 μM to 1 mM) may be used incombination with a light source that is used to initiate the reaction asdescribed herein. One example of a free thiol group may include, forexample, the amino acid cysteine, as described herein. Of course,peptides including a free cysteine or small molecules including a freethiol may also be used. Another example of a free radical transfer agentincludes N-Vinylpyrrolidone (NVP).

Alternatively, Michael and pseudo-Michael addition reactions, includingα,β-unsaturated carbonyl groups (e.g., acrylates, vinyl sulfones,maleimides, and the like) to a nucleophilic group (e.g., thiol, amine,aminoxy) may be used to anneal microgel particles 12 to form thescaffold 10. In another alternative embodiment, microgel particle 12formation chemistry allows for network formation through initiatedsol-gel transitions including fibrinogen to fibrin (via addition of thecatalytic enzyme thrombin).

Functionalities that allow for particle-particle annealing are includedeither during or after the formation of the microgel particles 12. Inone or more embodiments, these functionalities include α,β-unsaturatedcarbonyl groups that can be activated for annealing through eitherradical initiated reaction with α,β-unsaturated carbonyl groups onadjacent particles or Michael and pseudo-Michael addition reactions withnucleophilic functionalities that are either presented exogenously as amultifunctional linker between particles or as functional groups presenton adjacent particles. This method can use multiple microgel particle 12population types that when mixed form a scaffold 10. For example,microgel particle 12 of type X presenting, for example, nucleophilicsurface groups can be used with microgel particle 12 type Y presenting,for example, α,β-unsaturated carbonyl groups. In another embodiment,functionalities that participate in Click chemistry can be includedallowing for attachment either directly to adjacent microgel particles12 that present complimentary Click functionalities or via anexogenously presented multifunctional molecule that participates orinitiates (e.g., copper) Click reactions.

The annealing functionality can include any previously discussedfunctionality used for microgel crosslinking that is either orthogonalor similar (if potential reactive groups remain) in terms of itsinitiation conditions (e.g., temperature, light, pH) compared to theinitial crosslinking reaction. For example if the initial crosslinkingreaction consists of a Michael-addition reaction that is temperaturedependent, the subsequent annealing functionality can be initiatedthrough temperature or photoinitiation (e.g., Eosin Y, Irgacure®). Asanother example, the initial microgels may be photopolymerized at onewavelength of light (e.g., ultraviolent with Irgacure®), and annealingof the microgel particles 12 occurs at the same or another wavelength oflight (e.g., visible with Eosin Y) or vice versa. Besides annealing withcovalent coupling reactions, annealing moieties can include non-covalenthydrophobic, guest/host interactions (e.g., cyclodextrin), hybridizationbetween complementary nucleic acid sequences or nucleic acid mimics(e.g., protein nucleic acid) on adjoining microgel particles 12, orionic interactions. An example of an ionic interaction would consist ofalginate functionality on the microgel particle surfaces that areannealed with Ca2+. So-called “A+B” reactions can be used to annealmicrogel particles 12 as well. In this embodiment, two separate microgeltypes (type A and type B) are mixed in various ratios (between 0.01:1and 1:100 A:B) and the surface functionalities of type A react with typeB (and vice versa) to initiate annealing. These reaction types may fallunder any of the mechanisms listed herein.

In one embodiment, the microgel particles 12 are fabricated using eithermicrofluidic or millifluidic methods, generating deterministic microgelparticle length scales with small variability and in high throughput(e.g., frequencies greater than 10 particles/second). The coefficient ofvariation of the microgel particle 12 length scale (e.g., diameter) canbe within 35% or more preferably within 15% and even more preferablywithin 5% of the mean length scale. Milli- or microfluidics allow foruniform, pre-determined, concise material properties to be includedpre-, in-, and post-formation of microgel particles 12. Furthermore, themicrofluidic/millifluidic production mechanism allows for ease ofscaling-up production as well as good quality control over chemicalcomposition and physical characteristics of the microgel particles 12.The millifluidic and/or microfluidic technologies for microgel particle12 generation are easily scalable processes to create large amounts ofmaterial for commercial needs, while maintaining high accuracy andprecision in microgel particle 12 characteristics. Moreover, this is allaccomplished at low cost in comparison to other technologies involvingelectrospinning or large-scale fibrin purification.

In one embodiment, microgel particles 12 are formed using automatedfluidic methods relying on water-in-oil emulsion generation. Thisincludes microfluidic or millifluidic methods utilizing glass/PDMS,PDMS/PDMS, glass/glass, or molded/cast/embossed plastic chips to createwater in oil droplets with a size distribution variation that is lessthan 35%.

FIGS. 3A-3F illustrates one embodiment of a microfluidic device 20 thatis used to generate the microgel particles 12. The microfluidic device20 is formed in a substrate material 22 such as PDMS which may includeanother substrate material 24 (e.g., glass) that is bonded the substrate22. In this embodiment, the microfluidic device 20 includes a firstinlet 26, a second inlet 28, and a third inlet 30. As seen in FIG. 3A,the third inlet 30 is interposed between the first inlet 26 and thesecond inlet 28. In this embodiment, the first inlet 26 is coupled to asolution containing a 4-arm poly(ethylene glycol) vinyl sulfone (PEG-VS)backbone (20 kDa) that has been pre-modified with oligopeptides for celladhesive properties (e.g., RGD) and surface/tissue annealingfunctionalities (e.g., K and Q peptides). The PEG-VS backbone may beprefunctionalized with 500 μM K-peptide (Ac-FKGGERCG-NH₂ [SEQ ID NO: 1])(Genscript), 500 μM Q-peptide (Ac-NQEQVSPLGGERCG-NH₂ [SEQ ID NO: 2]),and 1 mM RGD (Ac-RGDSPGERCG-NH₂ [SEQ ID NO: 3]) (Genscript). Thesolution input to the first inlet 26 may contain about 5% (on a weightbasis) modified PEG-VS contained in a buffer of 0.3 M triethanolamine(Sigma), pH 8.25. The second inlet 28 is coupled to a solutioncontaining the crosslinker, which in one embodiment, is an 12 mMdi-cysteine modified Matrix Metallo-protease (MMP)(Ac-GCRDGPQGIWGQDRCG-NH₂ [SEQ ID NO: 4]) substrate (Genscript). Inexperiments conducted that utilized florescent imaging, the MMPsubstrate was pre-reacted with 10 μM Alexa-fluor 647-maleimide (LifeTechnologies). Of course, in practical applications, the use of thefluorescent probe is not needed. All solutions can be sterile filteredthrough a 0.2 μm Polyethersulfone (PES) membrane in a Luer-lock syringefilter.

As used herein, K-peptides refer to those peptides that contain thereina Factor XIIIa recognized lysine group. As used herein, Q-peptides referto those peptides that contain therein a Factor XIIIa recognizedglutamine group. Thus, peptide sequences beyond those specificallymentioned above may be used. The same applies to the RGD peptidesequence that is listed above.

The third inlet 30 is coupled to an aqueous solution containing 5% byweight of PEG-VS (unmodified by K, Q, or RGD peptides). The aqueousPEG-VS solution is preferably viscosity-matched with the PEG-VS solutionintroduced via the first inlet 26 and can be used to control the pH ofthe crosslinker solution and to inhibit crosslinking until dropletformation. By having the third inlet 30 interposed between the firstinlet 26 and the second inlet 28 the aqueous PEG-VS solution acts as abarrier that prevents any material diffusive mixing of reactivesolutions upstream of the droplet generation region. This significantlyincreases the lifespan of the device before fouling occurs. FIGS. 3E and3F illustrate how the inert liquid solution prevents mixing of left andright solutions prior to droplet segmentation. Note that the method ofmaking the microgel particles 12 will also work with omitting the thirdinlet 30, and adjusting peptide/crosslinker concentrations accordingly,yet the lifespan of the device will not be as long.

Referring to FIGS. 3A, 3B, and 3C, the first inlet 26, second inlet 28,and third inlet 30 are connected to, respectively, channels 32, 34, 36.The channels intersect at junction 38 and are carried in a commonchannel 40. The fourth inlet 42 is provided in the device and is coupledto an oil phase that contains a surfactant (e.g., 1% SPAN® 80 by volumealthough other surfactants can be used). The fourth inlet 42 isconnected to two channels 44, 46 that intersect at junction 48 at adownstream region of the common channel 40. The junction 48 in thedevice 20 is where the aqueous-based droplets are formed that includethe PEG-VS component and the crosslinker. The contents of the dropletsundergo mixing and will form the microgel particles 12 upon gelation,which in this embodiment is a function of the ambient temperature andthe passage of time. In this device, a fifth inlet 50 is provided thatis coupled to another oil phase that contains a surfactant at a highervolumetric percentage than that connected to the fourth inlet 42. Forexample, the fifth inlet 50 can be connected to an oil phase containing5% SPAN® 80 by volume. Again, other surfactants besides SPAN® 80 couldalso be used. The fifth inlet 50 is connected to two channels 52, 54that intersect at junction 56 in a pinching orientation as illustrated.

The common channel 40 continues to a series of progressively branchingbranch channels 58. The branch channels 58 permit continuous flow of themicrogel particles 12 through individual parallel channels where localenvironmental conditions can be optionally controlled. For example,temperature of the individual branch channels 58 can be controlled toregulate crosslinking conditions for the microgel particles 12.Likewise, the branch channels 58 may be illuminated with light tocontrol light-activated reactions. The microgel particles 12 may beremoved from the device 20 using the outlet 59. It should be understood,however, that regulation of the temperature of the branch channels 58 orthe use of light activation is entirely optional as the crosslinkingreaction may occur just through the passage of time when the device isoperated at or around ambient temperatures.

As best seen in FIG. 3D, the first inlet 26, second inlet 28, thirdinlet 30, fourth inlet 42, and fifth inlet 50 are connected,respectively, to fluid lines 26′, 28′, 30′, 42′, and 50′ that connect toa pumping device 51 or multiple pumping devices 51 that pumps respectivefluids into the correspondingly connected inlets 28, 28, 30, 42, 50. Thepumping device 51 may include separate pumps tied to each differentfluid. Examples of types of pumps that may be used include syringe pumpsor other pumps commonly used in connection with microfluidic devices. Inone aspect, the pumping device 51 uses regulated pressurized gas above afluid reservoir to pump fluid at the desired flow rate(s) through thedevice.

FIGS. 4A-4C illustrate an alternative embodiment of a microfluidicdevice 60 that is used to generate the microgel particles 12. In thisalternative embodiment, unlike the embodiment of FIGS. 3A-3C, there isno third inlet 30 that carries an aqueous solution that is used toseparate the PEG and crosslinking components prior to dropletgeneration. Rather, in this embodiment, the microfluidic device 60includes first inlet 62, a second inlet 64, a third inlet 66, and afourth inlet 68. The first inlet 62 is coupled to a modified PEG-VSsource such as that described above. The second inlet 64 is coupled to acrosslinking agent. The third inlet 66 is coupled to a source containingoil and a surfactant. The fourth inlet 68 is coupled to a sourcecontaining oil and a surfactant at a higher concentration than thatcoupled to the third inlet 66. In this embodiment, the first inlet 62and the second inlet 64 are coupled to respective channels 70, 72 thatlead to a common channel 74. The third inlet 66 is coupled to a pair ofchannels 76, 78 that intersect with the common channel 74 at a junction80 (best seen in FIG. 4B) where droplet generation occurs (droplets willform the microgel particles 12 upon reaction). The fourth inlet 68 iscoupled to a pair of channels 82, 84 that intersect with the commonchannel 74 at a downstream location 86 (best seen in FIG. 4B) withrespect to junction 80. As seen in FIG. 4A, the device 60 includes aseries of progressively branching branch channels 88 which are similarto those described in the context of the embodiment of FIGS. 3A-3C.Microgel particles 12 passing through branch channels 88 may collectedin a collection chamber 90 or the like which can be removed from thedevice 60. Fluid is delivered to the device 60 using fluid lines and apumping device as described previously in the context of the embodimentof FIGS. 3A-3C.

The fluidic conditions that lead to microgel particle 12 formationinclude, in one embodiment, on-chip mixing of a PEG-based andcrosslinker-based aqueous solutions, where one part contains basepolymer and the other contains the crosslinking or initiating agent. Ofcourse, in the embodiment of FIGS. 3A-3C, there is a three-input mixingwhich includes the aforementioned components plus the addition of theaqueous-based inert stream. These PEG and crosslinker solutions aremixed at either a 1:1 volumetric ratio, or another controllable ratio(controlled by relative flow rates into the device) up to 1:100. Theratios of the oil and total aqueous flow rates are controlled todetermine a specific size microgel particle 12, where these ratios canrange from 4:1 (aqueous:oil) down to 1:10 (aqueous:oil).

As explained above, in the embodiment of FIGS. 3A-3D, the chip device 20is designed to have three aqueous-based solutions combined to form themicrogel particles 12, wherein the base polymer andcrosslinking/initiating agent are separated by a non-reactive solutionupstream of the droplet generator to prevent reaction of solutions andfouling of the chip over time in the region upstream of dropletgeneration. In this configuration the portion of non-reactive solutionshould be equal to or less than base and cross-linker solutions, from 1to 0.05 times of the volume rate of the other solutions. This embodimentcan thus improve the reliability and lifetime of chips used for microgelgeneration. In addition, in this or the previous embodiment, cells canbe introduced into either of the two or three introduced aqueoussolutions to enable encapsulation of these cells (single cells orclusters of 2-20 cells per particle) within microgel particles 12 suchthat encapsulated cells can produce factors to enhance wound healing orcell ingrowth.

While FIGS. 3A-3D and 4A-4C illustrate different embodiments of amicrofluidic device 20, 60 that may be used to generate the microgelparticles 12, in an alternative embodiment, the microfluidic flow pathmay include a ‘T-junction’ architecture such as that illustrated in FIG.5. In this embodiment, the microfluidic device 92 includes a junctionformed between a first channel 94 that carries the aqueous phase while asecond channel 96 includes the oil phase. Droplets 97 are formed andcarried via an outlet channel 98 (which may be the same as the first orsecond channels 94, 96). Alternatively, different droplet formationconfigurations may be used to generate the microgel particles 12. Forexample, the device may generate droplets 97 using the gradient ofconfinement due to non-parallel top and bottom walls such as thatdisclosed in Dangla et al., Droplet microfluidics driven by gradients ofconfinement, Proc Natl Acad Sci USA, 110(3): 853-858 (2013), which isincorporated by reference herein.

In the microfluidic devices described above, the channel surfaces shouldbe modified such that the aqueous phase is non-wetting, which caninclude a fluorination of the surface, or converting the surfaces tobecome hydrophobic or fluorophilic, either by a covalent silane-basedtreatment or another non-specific adsorption based approach.Alternatively, a plastic polymer containing fluorophilic groupscomprises the chip material and can be combined with the previouslymentioned surface coatings or without a surface coating. Further, theoil used in the preferred embodiment should be either a mineral oil(paraffin oil) supplemented with a non-ionic surfactant, vegetable oilsupplemented with an ionic surfactant, or a fluorinated oil supplementedwith a fluorinated surfactant (or any combination of these twooil/surfactant systems). These microfluidic or millifluidic methodsgenerate monodisperse (coefficient of variation less than 35%)populations of microgel particles 12 in rates equal to or exceeding 10Hz, where collection is accomplished manually (by hand) or usingautomated fluidic handling systems. To prevent coalescence of microgelparticles 12 prior to completion of the crosslinking reaction sufficientsurfactant is necessary to stabilize the pre-gel droplets, however, highlevels of surfactant also destabilize the droplet generation process.Therefore, a preferred embodiment of the microfluidic system formicrogel particle 12 generation includes a low concentration ofsurfactant in the initial pinching oil flow (1% or less) that createsdroplets followed by addition of an oil+surfactant solution from aseparate inlet that is merged with the formed droplet and oil solutionand contains a higher level of surfactant (up to 10 times or even 50times higher than the initial surfactant). This is illustrated, forexample, in the embodiments of FIGS. 3A-3D and 4A-4C.

In another alternative embodiment, the two oil pinching flows have thesame concentration of surfactant. In still another embodiment, there isnot a second pinching oil flow, and only the flow-focusing oil flow togenerate droplets. Moreover, as explained above, in some alternativeembodiments, there is no second pinching oil flow and only thet-junction oil flow is used to generate droplets. Of course, thet-junction droplet junction may optionally be combined with a secondfocusing oil inlet with equal or greater surfactant concentration.

After formation, microgel particles 12 are extracted from the oil phaseusing either centrifugation through an aqueous phase, or filtrationthrough a solid membrane filtration device. For example, filtration maybe used to reduce the volume of free aqueous solution holding themicrogel particles 12 (free volume). In one embodiment, the aqueous freevolume is less than about 35% of the total volume. In anotherembodiment, for generation of intentionally polydisperse populations,microgel particle generation is carried out in a milli- or microfluidicplatform, generating stocks of relatively monodisperse microgelparticles 12 that are then mixed at desired ratios to obtaindeterministic distributions and ratios of microgel particle 12 sizes.Ratios of microgel particle 12 sizes can be controlled precisely tocontrol pore structure, or chemical properties in a final annealedscaffold 10 with stoichiometric ratios from: 1:1, 10:1, or exceeding100:1.

Alternatively, generation of microgel particles 12 via a water-in-oilsystem can also be carried out using sonic mixing methods or a rotatingvortex. These latter methods generate polydisperse populations ofmicrogel particles 12 with size ranges from 100 nanometers to 500micrometers. These particles can then be filtered using porous filters,microfluidic filtration, or other techniques known in the art to obtaina narrower size distribution of microgel particles 12 (e.g., coefficientof variation less than 50%). As another alternative, the componentmicrogel particles 12 of different shapes can be fabricated using stopflow lithography, continuous flow lithography, and other methods tocreate shaped particles that rely on shaping flows (see Amini et al.International Publication No. WO/2013/049404, which is incorporated byreference herein) combined with UV-initiated polymerization through ashape-defining mask. In this case the microgel particles 12 arenon-spherical with long and short dimensions that can vary between 5 and1000 micrometers. Shaped particles can also be fabricated by generatingspherical particles in a water in oil emulsion, followed by extrusion ofsaid particles through microfabricated constrictions that have lengthscales smaller than the diameter of the particle. The previouslyspherical particles adopt the shape of the constriction as theytransition to a gel and retain that shape as they gel in theconstriction by any of the crosslinker reactions listed above. The gelsretain that shape after exiting the microfabricated construction. Shapedparticles can allow for additional control of pores, overall porosity,tortuosity of pores, and improved adhesion within the final scaffoldformed by microgel particle 12 annealing.

In one or more embodiments, the microgel particles 12 are eithermodified covalently or not (e.g., inclusion spatially within bydiffusion) to provide biologically active molecules (e.g., smallmolecule drugs, antibiotics, peptides, proteins, steroids, matrixpolymers, growth factors, antigens, antibodies, etc.). Inclusion ofsignaling molecules after formation of the microgel particle 12 may beaccomplished through passive diffusion, surface immobilization(permanent or temporary), and/or bulk immobilization (permanent ortemporary).

In another embodiment, nanoparticles are included in the initialpre-polymer solution and incorporated in the microgel particles 12during initial polymerization or gelation, and the nanoparticles mayinclude biologically active molecules for sustained or rapid release anddelivery. In another embodiment, microgel particles 12 containing freeprimary amines (included as part ofa lysine-containing oligopeptides)can be modified with NHS-Azide. To this set of microgel particles 12 canbe added a protein modified with a NHS-phosphine, resulting insurface-coating of the microgel particles 12 with the modified protein.FIG. 10 illustrates an embodiment in which a microgel particle 12 hasnanoparticles embedded therein and a surface that has been modified witha protein using Click chemistry.

Following the production and optional modification, the microgelparticles 12 (which can be a homogeneous or heterogeneous mixture) maybe applied to a desired location (in vitro, in situ, in vivo). Thedesired location on mammalian tissue 102 can include, for example, awound site 100 or other site of damaged tissue. The microgel particles12 can be introduced alone in an aqueous isotonic saline solution orslurry (with preferably 30-99% volume fraction of microgel particles 12,and less preferably 1-30% volume fraction). Alternatively microgelparticles 12 can be introduced along with cells as single-cells oraggregates with cell to particle ratios from 10:1 to create dense cellnetworks within the final annealed scaffold 10 or 1:100 or even 1:1000to create sparsely seeded scaffolds 10 with cells that produce solublefactors useful for regeneration. In another embodiment microgelparticles 12 can be cultured with cells at a low volume fraction ofparticles (<10%) for a period of time in cell-permissive media topromote adhesion to the individual microgel particles 12. Thesecomposite cell-adhered microgel particles 12 can be introduced as theactive component that would anneal to form a microporous cell-seededscaffold 10, which may be beneficial to enhance the speed ofregenerative activity. Desired in vitro locations to introduce microgelparticles 12 include well plates (e.g., 6-well, 96-well, 384-well) ormicrofluidic devices to form 3D microporous culture environments forcells following annealing, and enable subsequent biological assays orhigh-throughput screening assays with more physiologically-relevant 3Dor multi-cellular conditions. For introduction in vitro, microgelparticle 12 solutions can be pipetted into wells or introduced viasyringe injection followed by introduction of an annealing solution ortriggering of annealing photochemically. Alternatively, a solution ofmicrogel particle 12 solution could be mixed with a slow actingannealing solution (annealing occurring over 10-30 min) before delivery.In situ locations include external wound sites (e.g., cuts, blisters,sores, pressure ulcers, venous ulcers, diabetic ulcers, chronic vascularulcers, donor skin graft sites, post-Moh's surgery sites, post-lasersurgery sites, podiatric wounds, wound dehiscence, abrasions,lacerations, second or third degree burns, radiation injury, skin tearsand draining wounds, etc.). Since the epidermis is an epithelialstructure, the microgel particle solution may be used to heal otherepithelial surfaces (i.e., urothelial (bladder and kidney),aerodigestive (lung, gastrointestinal), similarly to skin epithelium(i.e., stomach or duodenal ulcer; following penetrating trauma to thelung, bladder or intestinal fistulas, etc.). Additionally, the microgelparticle solution can be applied to other tissues through a catheter orcannula, such as nervous tissue and cardiac tissue where tissue ingrowthwould be beneficial to prevent scarring and to facilitate regenerativehealing following injury, such as after spinal cord trauma, cerebralinfarction/stroke, and myocardial infarction.

For introduction in situ microgel particle containing solution can bestored separately from an annealing solution and be mixed duringintroduction (a method analogous to epoxy adhesives) to preventpremature initiation of the annealing reaction before entry into a woundsite 100.

In another, the two solutions could be stored in a syringe orsqueeze-tube applicator with two barrels of equal or unequal diameters,such that when the plunger of the syringe is depressed or squeeze tubeis compressed it simultaneously delivers both the microgel particles 12and annealing solution at the correct stoichiometry. FIG. 6A illustratesone such embodiment of a delivery device 110 that includes a firstbarrel 112, a second barrel 114, and a plunger 116 that is used todispense the solution containing the microgel particles 12 from eachbarrel 112, 114. For example, the first barrel 112 contains microgelparticles 12 and thrombin at a concentration ranging from 0.1 to 5 U/mland the second barrel 114 contains the microgel particles 12 and FXIIIat a concentration of 0.1 to 1,000 U/ml). In both barrels 112, 114 thereis a 1 to 1 volume fraction of K and Q peptide containing microgelparticles 12 where the concentration of K and Q peptides range from10-1,000 μM in the microgel particles 12. In this embodiment, uponmixing the thrombin activates the FXIII (to form FXIIIa) and theresultant FXIIIa is responsible for surface annealing and linking of theK and Q peptides on the adjacent microgel particles 12.

Alternatively, the two barrels 112, 114 can contain two separatemicrogel particle 12 types with annealing moieties that require thecombination to initiate cross-linking. An alternative storage anddelivery method would be in a single barrel syringe 110 as illustratedin FIG. 6B or a multi-use or single-use compressible tube as illustratedin FIG. 6C (e.g., similar to toothpaste or antibiotic ointment) in whichthe microgel particle slurry can be squeezed out to a desired volume andspread over the wound site 100 and then annealed through exposure tolight, where the active agent for photochemistry is Eosin-Y at aconcentration of 100 μM although concentrations within the range of 10μM-1 mM will also work. Preferably, Eosin-Y is accompanied with aradical transfer agent which can be, for example, a chemical specieswith a free thiol group. An example of one such radical transfer agentincludes cysteine or peptides including cysteine(s) described herein(e.g., used at a concentration of 500 μM). The light should be deliveredvia a wide spectrum white light (incandescent or LED), or a green orblue LED light. A flashlight, wand, lamp, or even ambient light may beused to supply the white light. Exposure should occur between 0.1seconds and 1000 seconds, and the intensity of light should rangebetween 0.01 mW/cm² to 100 mW/cm² at the site of annealing. In anotherembodiment, light-mediated annealing can be accomplished using a UVlight (wavelengths between 300-450 nm), where the agent forphotochemistry is IRGACURE® 2959, at a concentration of 0.01% w/v to 10%w/v. The exposure time should be between 0.1 seconds and 100 seconds,with a light intensity of 0.1 mW/cm² to 100 mW/cm² at a site ofannealing. For embodiments in which light initiated annealing is used,microgel precursors 12 would be stored in opaque (opaque with respect towavelength range that initiates annealing) syringe or squeeze tubes 110containers prior to use. Desired in situ locations include internal cutsand tissue gaps (e.g., from surgical incisions or resections), burnwounds, radiation wounds and ulcers, or in cosmetic surgery applicationsto fill the tissue location and encourage tissue ingrowth andregeneration rather than the fibrotic processes common to contemporaryinjectables.

Delivery using double or single barrel syringes is also suited to thisindication as well as annealing using photoactivation and a UV or whitelight source that can be inserted into the surgical site. For both thein situ and in vivo applications the microgel particle slurry can bespread using a sterile applicator to be flush with the wound or moundedwithin and around the wound site 100 (within the wound and 2 mm to 1 cmbeyond the original wound extents) to create an annealed scaffold thatextends beyond the wound site 100 or tissue defect to provide additionalprotection, moisture, and structure to support tissue regeneration.

An annealing process is initiated through the application of a stimulus(e.g., radical initiator, enzyme, Michael addition, etc.) or throughinteractions with a stimulus that is already present at the site ofapplication of the microgel particles 12 that interacts with functionalgroups on the surface of the microgel particles 12, forming a solidcontiguous highly porous scaffold 10 formed from the annealed (linked)microgel particles 12. If used in tissue, the annealing process canallow for fusion of the scaffold 10 to the surrounding tissue, providingan effective seal, a local medication and/or cell delivery device, avascularized scaffold for in vivo sensing, and a better path to tissueregeneration. The annealing process allows for on-site/on-demand gelformation (which is ideal for in vitro and in vivo applications), forexample delivery through a small incision to a minimally-invasivesurgical site or through injection by a needle or through a catheter orcannula. The scaffold 12 may comprise of homogeneous or heterogeneouspopulations of microgel particles 12. As discussed, the heterogeneouspopulations of microgel particles 12 may vary in physical (e.g., insize, shape, or stiffness) or vary in chemical composition (e.g., variedratios of degradable linkers, or L- or D-amino acids to modifydegradation rate, varied annealing moieties, cell adhesive moieties, orloading of microgels 12 with bioactive molecules or nanoparticles). Theheterogeneous composition of the final annealed scaffold 10 can berandom or structured in layers of uniform composition to creategradients in micro-porous structures (by varying microgel particle 12sizes in layers, for example) or gradients of chemical composition (bylayers of microgel particles 12 with different composition or bio-activemolecule loading). Gradients may be useful in directing cell ingrowthand tissue regeneration in vivo, or development of tissue structures invitro. Gradients in microgel particle 12 composition could be achievedby delivering sequential slurries of a gel of a single composition,followed by annealing, and then subsequent delivery of the next gel of asecond composition, followed by annealing which links the new layer ofmicrogels to the previous layer, until a desired number of layers havebeen accumulated. The thickness of each layer can be controlled usingthe volume of slurry injected and area of the injection site. Analternative embodiment to achieve gradients is to load a multi-barrelsyringe applicator such as that illustrated in FIG. 6A with differentmicrogel compositions in each of the barrels. Each of the barrels aresimultaneously compressed and feed to the nozzle 120 in layered sheets.The nozzle 120 itself of the syringe applicator can be non-circular orrectangular to create a layered slurry of multiple composition that isinjected to a site in a ribbon-like structure, which can then beannealed in this arrangement. Formation of the structurally contiguousannealed scaffold 10 may be achieved through radical, enzymatic orchemical (e.g., Click chemistry) processes.

In one or more embodiments, annealing occurs through surface chemistryinteractions between microgel particles 12 once they are ready to beplaced at the delivery site. In one embodiment, the process occursthrough radical-initiated annealing via surface polymerizable groups(e.g., radical initiation by photo-sensitive radical initiators, etc.).In another embodiment, the process occurs through enzymatic chemistryvia surface presented enzymatically-active substrates (e.g.,transglutaminase enzymes like Factor XIIIa). In another embodiment, theprocess occurs through covalent coupling via Michael and pseudo-Michaeladdition reactions. This method can use multiple microgel particlepopulation types that when mixed form a solid scaffold 10 (e.g.,microgel particle 12 type A presenting, for example, nucleophilicsurface groups and microgel particle 12 type B presenting, for example,α,β-unsaturated carbonyl groups). In another embodiment, the processoccurs through Click chemistry attachment. Similarly, this method canuse heterogeneous microgel particle 12 populations that when mixed forma solid microporous gel. In another embodiment, annealing may beachieved using light (for example, either white light or UV light) toinitiate a chemical reaction between molecules on the gel surfaces,mediated by a light activated molecule in solution in and around (ordirectly covalently liked to) the microgels as described herein.

In one preferred embodiment, the microgel particles 12 include a PEGbased polymeric backbone in combination with an enzymatically degradablecrosslinker to allow for bioresorbability. In certain embodiments, thePEG-based polymeric backbone is a 4-arm poly(ethylene glycol) vinylsulfone (PEG-VS) backbone pre-modified with oligopeptides for celladhesive properties (e.g., RGD) and surface annealing functionalities(e.g., K and Q peptides) and the cross-linker is a matrixmetalloprotease (MMP)-degradable cross-linker.

In one or more embodiments, microgel particles 12 are formed by awater-in-oil emulsion. Gelation of the microgel particles 12 occurs uponcombination of PEG solution with cross-linker solution (followed shortlyby partitioning into microgel droplets before completion of gelation). Avariety of substrates, including peptide ligands, can be further addedfor enhanced bioactivity. In one embodiment, scaffold formation isaccomplished by addition and activation of radical photo-initiator tothe purified microgel particles 12 to induce chemical cross-linking. Inanother embodiment, scaffold formation is accomplished by the use and/oractivation of an endogenously present or exogenously appliedtransglutaminase enzyme, Factor XIII, to the purified microgel particles12 that have been modified with two peptide ligands eitherpre-formation, during formation, or post-formation to induce enzymaticcross-linking. In a separate embodiment, scaffold formation isaccomplished using a combination of the aforementioned radical andenzymatic methods.

The resultant scaffold 10 of the presently disclosed subject matterprovides advantages over current porous scaffold technologies due to theability to form a fully interconnected microporous scaffold in vivo. Ingeneral, porous scaffolds provide for greater access for live cells dueto the freedom of movement through the pores (i.e., not requiringdegradation to allow penetration like all current and previousnon-porous and nano-porous scaffolds). For example when implanting andannealing a scaffold 10 in a skin wound in vivo, significantly enhancedcell invasion and tissue-structure in growth was observed after 5 dayswhen compared to a non-porous gel of the same material as seen in FIG.7B. FIG. 7A illustrates H&E staining of tissue sections in SKH1-Hr^(hr)mice for tissue injected with the scaffold 10 (identified as MAPscaffold) as well as the non-porous control 24 hours after injection.FIG. 7B illustrates a graph of wound closure (%) as a function of dayspost-injection. This graphs shows that over a five (5) day period thereis statistically significant improvement in the wound closure rates forusing the scaffolds 10 when compared to non-porous bi-lateral controls(N=5). FIG. 7C illustrate representative images of wound closure duringa 5 day in vivo wound healing model in SKH1-Hr^(hr) mice. FIG. 7Dillustrates representative images of wound closure during 7 day in vivoBALB/c mice experiments. FIG. 7E illustrates wound closurequantification data from BALB/c in vivo wound healing. After 7 days invivo, the scaffolds 10 promote significantly faster wound healing thanthe no treatment control, the non-porous PEG gel, and the gels lackingthe K and Q peptides. Porous gels created ex vivo to precisely match thewound shape using the canonical, porogen-based, casting method showedappreciable wound healing rates, comparable to the scaffolds 10, butlacking injectability (N≧5). FIG. 7F illustrates traces of wound bedclosure during 7 days in vivo for each treatment category correspondingto FIG. 7D.

Furthermore, therapeutic agents applied to the microgel particles 12 orthe scaffold 10 can be released slowly or rapidly, and the scaffold 10has the ability to break down over a pre-determined period of timeeither from hydrolysis, proteolysis, or enzymolysis, depending on theintended treatment (e.g., if it is being used to treat a chronic wound,a more stable cross-linker that degrades slowly over time is used).Additionally, the annealing quality of the microgel scaffold 10 allowsthe scaffold 10 to function as a tissue sealant (e.g., acute wounds,surgical closure, etc.), and the filling of different molded shapes thatare clinically useful to mimic tissues. FIG. 7G illustrates how themicrogel particle containing solution or slurry can be applied using asyringe device like that of FIG. 6A or 6B into a treatment site wherethe microgel conforms to the shape of the injection site (e.g., in thiscase a star-shaped site) and subsequent annealing of the scaffold 10into the star shape.

By adjusting the rate of degradation of the microgel scaffolds 10 thescar forming or regenerative response in a wound can be modified. In oneembodiment, the degradation rate of the microgel scaffolds 10 wasmodified by using D- instead of L-amino acids in the MMP-degradablecrosslinker. Adjusting the ratio of microgel particles 12 with D- orL-chirality in the crosslinker adjusted the rate of degradation in thetissue. Scaffolds 10 made from mixtures of D and L crosslinked microgels(at a 1:1 ratio) resulted in gels present in the tissue 21 days afterinjection, however in the D-only gels, there was no remaining gel leftafter 21 days in vivo. Tissue healing and scarring response also dependson the stoichiometry of D:L, and thus the degradation rate. FIGS. 8A-8Gshow the effects of scar reduction when using a 1:1 mixture of D:L, ascompared directly to a no treatment wound. Dermal thickness is doubledand scar size is reduced by 25% in the 1:1 D:L gel treatment.Additionally, six (6) times more hair follicles and sweat glands arepresent in the gel-treated case, compared to the no treatment case.

EXPERIMENTAL

A microfluidic water-in-oil emulsion approach was used to segment acontinuous pre-gel aqueous phase into uniform scaffold building blocksas described herein. Generating microgel particles 12 as building blocksserially at the microscale, rather than using the typical vortex andsonication-based approaches allowed tight control over the formationenvironment and ultimate material properties of the emergent scaffold10. By tuning the flow rates of both the pre-gel solution and thepinching oil flow, as well as the geometry of the microfluidic channel,a range of microgel particle sizes were created with low polydispersity.Although the fabrication method was serial, it retained practicality inits high throughput nature, with generation rates that ranged from 250Hz for larger particles (>100 μm) to ˜1200 Hz for small particles (˜15μm). This translated to roughly 100 μl of pre-swollen gel every 50 minfor a single device. This approach ultimately resulted in particles thatwere highly monodisperse, both physically and chemically. Microfluidicgeneration of microgel particle “building blocks” is a readily scalableprocess: a practical requirement for wide adoption and use.

The resultant microgel particles 12 were composed of a completelysynthetic hydrogel mesh of poly(ethylene)glycol-vinyl sulfone (PEG-VS)backbones decorated with cell-adhesive peptide (RGD [SEQ ID NO: 3]) andtwo transglutaminase peptide substrates (K [SEQ ID NO: 1] and Q [SEQ IDNO: 2]). The microgel particles 12 were crosslinked via Michael typeaddition with cysteine-terminated matrix metalloprotease-sensitivepeptide sequences that allowed for cell-controlled material degradationand subsequent resorption.

The microgel particles 12 were purified into an aqueous solution ofisotonic cell culture media for storage and when used to form a gel wereannealed to one another via a non-canonical amide linkage between the Kand Q peptides mediated by activated Factor XIII (FXIIIa), a naturallyoccurring enzyme responsible for stabilizing blood clots. Thisenzyme-mediated annealing process, allowed incorporation of living cellsinto a dynamically forming scaffold 10 that contained interconnectedmicroporous networks. Following addition of FXIIIa, but prior toscaffold annealing, a slurry of the microgel particles 12 can bedelivered via syringe application, ultimately solidifying in the shapeof the cavity in which they are injected. FIG. 9A illustrates how theannealing kinetics can be altered by the adjustment of pH andtemperature. The annealing environment chosen for this experiment was pH8.25 and a temperature of 37° C.

Structural changes leading to over a three-fold increase in storagemodulus in the annealed gels was observed upon addition of FXIIIa to themicrogel particles 12. Annealing was confirmed as being necessary forscaffold formation via high-vacuum SEM observation, wherein upondehydration the scaffolds adopted a highly stretched but interconnectedmesh whereas building blocks without FXIIIa separated into individualspherical beads (FIG. 9E).

By tuning the microgel particle size and composition a diverse set ofassembled scaffolds 10 were able to be generated. By using microgelparticles 12 from 30 to 150 μm in diameter, networks with median poresdiameters ranging from ˜10 to ˜35 μm were achieved). Different PEGweight percentages and crosslinker stoichiometries were screened todemonstrate a range of easily achievable storage moduli from ˜10 to 1000Pa that spans the stiffness regime necessary for mammalian soft tissuemimetics. FIG. 9B illustrates different hydrogel weight percentages wereused to produce different stiffness materials. FIG. 9C illustratesdifferent crosslinker stoichiometries (r-ratio of crosslinker ends (−SH)to vinyl groups (−VS)) that were used to produce different stiffnessvalues in the resultant gel. FIG. 9D illustrates a graph of the %degradation as a function of time for both the non-porous control aswell as the inventive porous gel described herein. Degradation kineticsof particle-based, porous gel and the non-porous are shown for equalvolumes of gels in vitro. The particle-based, porous gels degrade fasterthan non-porous gel due to higher surface area to volume ratios andfaster transport through the microporous gel. Degradation was carriedout using 1:1000 TrypLE®, resulting in higher protease concentrationsthan in a wound bed and faster degradation kinetics. FIG. 9E illustratesSEM images of a scaffold annealed with FXIIIa. FIG. 9F illustrates SEMimages of microgel particles 12 without FXIIIa. Un-annealed particlesare seen in FIG. 9F.

In order to assess the ability of the generated scaffold to support cellgrowth and network formation, an in vitro cell morphology andproliferation model using three human cell lines was developed. Theseincluded: Dermal Fibroblasts (HDF), Adipose-derived Mesenchymal StemCells (AhMSC), and Bone Marrow-derived Mesenchymal Stem Cells (BMhMSC).A single-cell suspension was dynamically incorporated within a FXIIIaannealed gel. The three cell lines exhibited high cell viability (≧93%)following twenty-four (24) hours of culture within the scaffold. The HDFand AhMSC cell lines demonstrated continued proliferation over a six-dayculture period with doubling times of 1.5 and 2 days, respectively.BMhMSCs were observed to undergo proliferation as well, however with anextended calculated doubling time of ˜12 days.

Cells incorporated into the scaffold began to exhibit spread morphology90 minutes following the onset of annealing. After two (2) days inculture, all observed cells within the scaffolds exhibited a completelyspread morphology, which continued through day six (6). Importantly, anextensive network formation for all cell lines was observed by day two(2). Cell networks increased in size and complexity through the entiretyof the experiment. The BMhMSCs were of particular note, as theirexpansive network formation and slower proliferation rate indicated thatthese cells were able to spread to extreme lengths, forming highlyinterconnected cellular networks within the microporous scaffolds.Notably, cells that were grown in non-porous gels of identical chemicalproperties (5 wt %, G′=600 Pa gel) and mechanical properties (4.5 wt %,G′=350 Pa gel) maintained viability but did not exhibit any appreciablenetwork formation, even after six days in culture.

It was hypothesized that the ability of the scaffolds to enable bothcell proliferation and expedient network formation in vitro wasindicative of an ability to support in vivo cell migration and bulktissue integration within the scaffold. To test this hypothesis, amurine skin wound healing model was used, addressing a tissue ofinterest for previous implanted porous biomaterials. Importantly, woundcontraction was prevented using a sutured rubber splint that limitedclosure to tissue ingrowth, better simulating the human healingresponse. Because of the injectability of the microgel particle-basedscaffold, the microgel particles were able to be directly delivered tothe wound site, followed by in situ annealing via exogenous FXIIIa. Thisprovided a seamless interface by simultaneously linking the microgelparticle “building blocks” to one another as well as to endogenouslysine and glutamine residues present in the surrounding tissue.Similarly, a seamless interface was observed for the chemicallyidentical, nonporous bi-lateral control. Despite their similarinterface, the generated scaffold resulted in significantly faster woundclosure than the non-porous controls (60% versus 100% remaining woundarea after 5 days, respectively) when injected into the wounds ofCLR:SKH1-Hr^(hr) mice as seen in FIG. 7B.

The disparities in wound closure rates led to the investigation of thedifferences in tissue responses to the non-porous and injectablepartible-based gel. The scaffold injection using the microgel particlesresulted in extensive wound re-epithelialization after five (5) days invivo. keratin-5⁺ cells were observed with stratified squamous morphologyover the apical surface of the scaffold, however no cells (keratin-5⁺ orotherwise) were observed past the non-porous wound edge. Importantly,the scaffold was able to sustain the formation of what appeared to be acomplete hair follicle with adjoining sebaceous gland within the woundbed resembling the structure of these glands in the uninjured skin.Further, other instances of large Keratin-5⁺ tissue structures wereobserved within the scaffold including tubular structures and epithelialinvaginations. It is hypothesized that together, these results are anindication of higher order collective migration (i.e., movement ofmulticellular clusters in concert) contributing to epidermalregeneration. Although cells were able to infiltrate the non-porousbi-lateral controls (as indicated by DAPI staining), no evidence ofre-epithelialization or cutaneous tissue formation was found after five(5) days in vivo.

Through further investigation, it was found that the scaffold promotedbulk integration via complex vascular network formation in vivo. Afterfive (5) days, both endothelial cells and supporting pericytes werepresent within the scaffold, while only single branches of endothelialcells without supporting pericytes were present in the non-porousbilateral controls. The presence of co-localized endothelial cells andpericytes was evidence of mature vessel network formation. To ourknowledge, this is the first instance of early (<7 days) pericytemigration into a synthetic injectable material or implanted porousscaffold without the inclusion of exogenous growth factors.

While investigating the seamless interface provided by the injectablescaffolds differences were observed in both overall and immune cellquantities at day one (1). After one (1) day post-injection, thescaffolds contained significantly higher numbers of cells within thescaffold than their non-porous bi-lateral controls. This corroboratedthe greater ease of cell mobility previously observed in our in vitronetwork formation experiments. Further, the scaffold and its surroundingtissue contained a significantly lower number of polymorphonuclear cellswhen compared to the non-porous bi-lateral control of the same mouse.This result indicated an overall lower initial innate immune response tothe scaffolds at day one (1). After five (5) days post-injection, lowerfractions of CD11b⁺ cells (activated leukocytes) were present both inthe surrounding tissue and within the scaffold relative to thenon-porous controls, indicating a sustained lower level of inflammatoryimmune response, in agreement with what has been observed in ex vivoconstructed and implanted micro-porous scaffolds. Combined, these tworesults support a presently underexplored geometric component to immunestimulation from chemically-identical injectable biomaterials.

The annealed, microgel particle-based scaffolds represent a new class ofinjectable biomaterial that introduces microscale interconnectedporosity through robustly achieved imperfect self-assembly and annealingof individual building blocks. This approach allows control ofmicro-scale and hierarchical macro-scale properties throughdeterministic chemical composition and microfluidic particle generation.Both incorporated live cells and surrounding host tissue are able toimmediately infiltrate the scaffold without the need for materialdegradation, a feat never before accomplished using injectablescaffolds.

In vivo, the injectable microgel particles completely filled the tissuevoid, providing a seamless boundary with the surrounding tissue. Theinterconnected microporosity of the resulting scaffold promoted cellularmigration at the wound site that resulted in greater bulk integrationwith the surrounding tissue while eliciting a reduced host immuneresponse, in comparison to an injectable non-porous control. Ultimatelythis led to faster healthy tissue reformation than with similarlycomprised injectable non-porous gels.

This gel system presents a fundamental change in the approach tobottom-up modular biomaterials by utilizing the negative space oflattice formation to promote the development of complexthree-dimensional networks on time scales previously unseen usingcurrent hydrogel technologies. The “plug and play” nature of thisstrategy allows the incorporation of a wide range of already establishedmaterials (e.g., fibrin), signals (e.g., growth factors), and cellpopulations (e.g., stem cells). Complex combinations of building blockswith deterministic chemical and physical properties may enable tissueregeneration in a range of distinct physiological niches (e.g., neural,cardiac, skin, etc.), where particle-annealed scaffolds are tailored toeach niche via their building block properties. The unique combinationof microporosity, injectability, and modular assembly inherent toscaffolds has the potential to alter the landscape of tissueregeneration in vivo and tissue creation de novo.

Microfluidic water-in-oil droplet generators were fabricated using softlithography as previously described. Briefly, master molds werefabricated on mechanical grade silicon wafers (University wafer) usingKMPR 1025 or 1050 photoresist (Microchem). Varying channel heights wereobtained by spinning photoresist at different speeds, per themanufacturer's suggestions. Devices were molded from the masters usingpoly(dimethyl)siloxane (PDMS) SYLGARD® 184 kit (Dow Corning). The baseand crosslinker were mixed at a 10:1 mass ratio, poured over the mold,and degassed prior to curing for 6 hours at 65° C. Channels were sealedby treating the PDMS mold and a glass microscope slide (VWR) with oxygenplasma at 500 mTorr and 75 W for 15 seconds. Immediately after channelsealing, the channels were functionalized by injecting 100 μl of asolution of RAIN-X® and reacting for 20 minutes at room temperature. Thechannels were then dried by air followed by desiccation overnight.

Droplets were generated using a microfluidic water-in-oil segmentationsystem as illustrated in FIGS. 3A-3F and 4A-4C. The aqueous phase is a1:1 volume mixture of two parts: (i) a 10% w/v 4 arm PEG-VS (20 kDa) in300 mM triethanolamine (Sigma), pH 8.25, prefunctionalized with 500 μMK-peptide (Ac-FKGGERCG-NH₂ [SEQ ID NO: l]) (Genscript), 500 μM Q-peptide(Ac-NQEQVSPLGGERCG-NH₂ [SEQ ID NO: 2]), and 1 mM RGD (Ac-RGDSPGERCG-NH₂[SEQ ID NO: 3]) (Genscript) and (ii) an 8 mM (12 mM for the three-inletdevice) di-cysteine modified Matrix Metallo-protease (MMP)(Ac-GCRDGPQGIWGQDRCG-NH₂ [SEQ ID NO: 4]) (Genscript) substratepre-reacted with 10 μM Alexa-fluor 647-maleimide (Life Technologies).All solutions were sterile-filtered through a 0.2 μm Polyethersulfone(PES) membrane in a Leur-Lok syringe filter prior to use in thesegmentation system.

Generation was performed at 37° C. on an incubated microscope stage(NIKON® Eclipse Ti) for real time monitoring of microgel quality. Theinput aqueous solutions did not appreciably mix until dropletsegmentation (Peclet number >10). The oil phase was a heavy mineral(Fisher) oil supplemented with 0.25% v/v SPAN® 80 (Sigma-Aldrich).Downstream of the segmentation region, a second oil inlet with a highconcentration of SPAN® 80 (5% v/v) was added and mixed to the flowingdroplet emulsion. Ultimately, the microgel-in-oil mixture exited into alarge (12 mm diameter, ˜1 mL volume) well, where the microgel particlescured at 37° C. for a minimum of 1 hour. The mixture was then extractedand purified by overlaying the oil solution onto an aqueous buffer ofHEPES buffered saline pH 7.4 and pelleting in a table top centrifuge at18000×g for 5 mins. The microgel-based pellet was washed in HEPESbuffered saline pH 7.4 with 10 mM CaCl₂ and 0.01% w/v Pluronic F-127(Sigma). The microgel aqueous solution was then allowed to swell andequilibrate with buffer for at least 2 hours at 37° C.

To determine the operational regime of droplet segmentation, deviceoperation was monitored in real time using a high-speed camera(Phantom), followed by image analysis for size and polydispersitymeasurement (using ImageJ software) as well as segmentation frequency(Phantom PC2). For stable droplet segmentation on this platform: (i)initiate all flows simultaneously (both aqueous flows and both oilflows) at 5 μl/min until all air has been flushed from the device, (ii)turn down aqueous flow rates to the desired overall volumetric rate(aqueous flow rate between 1.5 and 2 μL/minute and oil flow ratesbetween 1 and 5 μL/minute for 5 minutes, (iii) aspirate all accumulatedliquid from collection well to ensure collection of monodisperse μgels,and (iv) run generation.

Fully swollen and equilibrated “building block” microgel particles werepelleted by centrifugation at 18000×g for five minutes, and the excessbuffer (HEPES pH 7.4+10 mM CaCl₂) was removed by aspiration and dryingwith a cleanroom wipe. Subsequently, microgel particles were split intoaliquots, each containing 50 μl of concentrated building blocks. Anequal volume of HEPES pH 7.4+10 mM CaCl₂ was added to the concentratedbuilding block solutions. Half of these include Thrombin (Sigma) to afinal concentration of 2 U/ml and the other half includes FXIII (CSLBehring) to a final concentration of 10 U/ml. These solutions were thenwell mixed and spun down at 18000×g, followed by removal of excessliquid with a cleanroom wipe (American Cleanstat).

Annealing was initiated by mixing equal volumes of the building blocksolutions containing Thrombin and FXIII using a positive displacementpipet (Gilson). These solutions were well mixed by pipetting up anddown, repeatedly, in conjunction with stirring using the pipet tip. Themixed solution was then pipetted into the desired location (mold, wellplate, mouse wound, etc.).

To determine the gelation kinetics for each microgel, a macroscale (50μL) non-porous gel was generated with the same chemical composition. A30 μL solution of 2× PEG-VS+peptides (RGD, K, and Q peptides) dissolvedin 0.3 M TEOA was combined with 30 μL of 2× MMP-1 crosslinker dissolvedin water. The mixture was quickly vortexed and 50 μL of the mixture wasplaced between two 8 mm rheological discs at a spacing of 1 mm (AntonPaar Physica MCR301 Rheometer). The storage modulus was then measuredover a period of 20 minutes (2.5 Hz, 0.1% strain).

To determine the bulk storage modulus of the pre-annealed microgelparticles and post-annealed scaffold an amplitude sweep (0.01-10%strain) was performed to find the linear amplitude range for each. Anamplitude within the linear range was chosen to run a frequency sweep(0.5-5 Hz). For pre-annealed microgel particles, 50 μL of microgelparticles (5 wt % PEG-VS 4-arm MW=20 KDa, r=0.8 MMP-1 crosslinker, withsynthetic peptide concentrations of 250 μM synthetic K, 250 μM syntheticQ, 500 μM synthetic RGD) was injected between two 8 mm rheological discsat a spacing of 1 mm. For post-annealed scaffold measurement, we firstpipetted 50 μL of microgel particles (N=3) (5 wt % PEG-VS 4-arm MW=20KDa, r=0.8 MMP-1 crosslinker, with synthetic peptide concentrations of250 μM synthetic K, 250 μM synthetic Q, 500 μM synthetic RGD) spikedwith FXIIIa, 5 U/mL final concentration, and thrombin, 1 U/mL finalconcentration, between two glass slides. This mixture was allowed topartially anneal for 10 minutes before removal of top glass slide andplacement in a humidified incubator at 37° C. for 90 minutes. Thescaffolds were then placed into HEPES buffered saline (pH 7.4) overnightto reach equilibrium. The samples were then placed between two 8 mmdiscs on the rheometer and tested identically to the pre-annealedmicrogel particles.

To determine median pore size in the annealed microgel scaffolds, stocksolutions of different sized microgel particles were used to annealthree separate scaffolds from each (9 scaffolds in total), as describedabove. Using a Nikon Ti eclipse equipped with the C2 laser LED confocal,individual slices were taken in each gel, separated by 50 m between eachslice (10 slices per gel, with 30 total slices for each gel type). Theseimages were then analyzed using a custom script written in MATLAB®, toidentify the pore regions and calculate each one's size in px². Eachindividual pore's size was then used to calculate the median pore sizefor that gel, and converted to μm² using the pixel to μm conversion fromthe original microscope image (0.31 μm/px). These areas were thenconverted to a characteristic length measurement by forcing the areas toa circle, and calculating the characteristic diameter of these circles.For 30 μm microgel particles, mean pore diameter was around 12 μm. For100 μm microgel particles, mean pore diameter was around 19 μm. For 150μm microgel particles, mean pore diameter was around 37 μm. Note thatthe interstices or voids are continuous and not similar to thewell-defined spherical open regions connected by circular pores asproduced through microparticle leaching or inverse opal gel fabricationmethods, however, referring to a pore diameter is useful to simplydescribe the length scale of the void spaces.

To determine if microgel particles were covalently linked after additionof FXIIIa, SEM was used to directly visualize scaffolds. Microgelparticle mixtures were either treated with FXIIIa (10 U/ml) or withbuffer only. Subsequently, the building block solutions were placed ontoa 1×1 in silicon wafer piece, and dried in an SEM (Hitachi S4700) highvac chamber (1×10⁻³ mTorr). Building blocks with or without FXIIIa werethen visualized using 10 kV (10 mA max) on either 200× or 500× as seenin FIGS. 9D and 9F.

HEK293T cells constitutively expressing GFP via lentiviral transfectionwere maintained in DMEM (Life Technologies) supplemented with 10 μg/mlpuromycin. Three cell lines were used for in vitro experiments: humandermal fibroblasts (HDF, Life Technologies), bone marrow-derived humanmesenchymal stem cells (BMhMSC, Life Technologies), and adipose-derivedhuman mesenchymal stem cells (AhMSC, Life Technologies). All cell lineswere maintained according to manufacturer's specifications (before andafter incorporation into porous or non-porous gels). Specifically, forthe MSC populations reduced-serum, basal medium (Life Technologies) wasused to retain stemness.

For quantification of cell proliferation and visualizations of networkformation in the porous scaffolds in vitro, particle-based scaffoldswere annealed with microgel particles as described above, with theaddition of cell suspensions to the building block solutions prior toannealing. For each cell line, cell suspensions were prepared at a finalconcentration of 25×10⁶ cells/ml in respective culture mediaun-supplemented with serum. Subsequently, 2 μl of cell suspension wasadded to 50 μl of microgel particle mixture containing FXIII andcombined with 50 μl of microgel particle mixture containing Thrombin(500 cells/μl of gel). This mixture was injected into the corner of acoverslip-bottom PDMS well. The well top was covered with a secondcoverslip and the μgel/cell mixture was allowed to undergo annealing for90 minutes at 37° C.

After annealing was completed, the top coverslip was removed, and theappropriate complete culture media was added to the PDMS well. For theday 0 time point, 4% PFA was added directly to the PDMS wells andallowed to fix overnight at 4° C. Other cells were grown in 5% CO₂ and37° C. for the times indicated (2, 4, and 6 days), at which point theywere washed once with 1×PBS and fixed with 4% PFA overnight at 4° C.HEK-293-T cells were incorporated into a star-shaped mold by mixingcells with microgel particles (as described above) and pipetting 5 μl ofthe mixture into the center of the mold. Immediately following, microgelparticles without cells were pipetted in the remainder of the mold, andannealed as described above.

Proliferation was assessed by counting the number of cell nuclei presentin the particle-based scaffold constructs after 0, 2, 4, and 6 days ofculture in vitro. Nuclei were stained with a 2 μg/ml DAPI solution in1×PBS for 2 hours, followed by visualization on a Nikon C2 using the 405nm LED laser. Specifically, each scaffold was imaged by taking 55 zslices in a 150 μm total z height and compressing every 5 slices into amaximum intensity projection (MIP) image. Nuclei in the MIPs wereenumerated using a custom MATLAB® script, counting the total number ofcells. For each time point, z-stack images of three separate microgelscaffolds were analyzed, where each z-stack image measured a totalvolume of 1270×1270×150 μm³ (or ˜280 nL). The 90 minute counts lead to acalculation of ˜525 cells/μl of gel, consistent with the experimentalamount added (500 cells/μl of gel).

For visualization of cell network formation within the microgelscaffolds in vitro, the constructs were prepared, grown, and fixed asabove. The scaffolds were blocked with 1% BSA in 1×PBS for 1 hour atroom temperature, followed by staining for f-actin via a Rhodamine-Bconjugate of phalloidin (Life Technologies) for 3 hours at roomtemperature. The scaffolds were then washed with 1% BSA in 1×PBS,followed by counterstaining with a 2 μg/ml DAPI solution in 1×PBS for 1hour at room temperature. Imaging was performed as with proliferationimaging, with the exception of using a 40× magnification water immersionlens. Total heights of image stacks were 130 μm, with the total numberof slices at 260 (volume captures ˜15 nL).

PEG-VS scaffolds (5 wt % PEG-VS 4-arm MW=20 KDa, r=0.8 MMP-1crosslinker, with synthetic peptide concentrations of 250 μM synthetic K[SEQ ID NO: 1], 250 μM synthetic Q [SEQ ID NO: 2], 500 μM synthetic RGD[SEQ ID NO: 3]) were used to encapsulate cells (500 cells/μL). Celllines used were the same as in microgel scaffold experiments. Gels wereformed for 20 minutes (TEOA 0.3 M, pH 8.25) before being placed intoappropriate media. The gels were fixed after pre-determined time points(t=90 minutes, 2 days, 4 days, and 6 days) using PFA overnight at 4° C.,washed and stored in PBS. Gels were stained as in the microgelscaffolds. All samples were stored at 4° C. in PBS with P/S when notbeing imaged. Imaging was performed using a NIKON® C2 confocal exactlyas in the microgel scaffold in vitro experiments.

CLR:SKH1-Hrhr Mice (Charles River Laboratories) (N=6 per test) wereanesthetized with isofluorane (1.5% for 10 minutes), followed byclipping of nails and injection of painkiller (buprenorphine, 60 μL per20 g at 0.015 μg/μL). The skin was pulled taut and a 4 mm biopsy punchwas used to create identical circular wounds on the back of the mouse.The periphery of the wounds was secured using a rubber splint sewn via7-8 stitches to the surrounding skin to prevent wound closure bycontraction. Either non-porous or porous hydrogel including 10 U/mlFXIIIa was injected into wound beds, allowed to undergo gelation for 10minutes, followed by subsequent covering of the wound by a stretchygauze wrap to prevent animal interaction. The mice were then separatedinto individual cages. Pain medication was administered subcutaneouslyevery 12 hours for the next 48 hours (for Day 1 sacrifices pain killerwas administered once after surgery).

At Day 1, mice (N=6) were sacrificed via isofluorane overdosing,followed by subsequent spinal dislocation. The skin of the back wasremoved using surgical scissors and the wound site was isolated via a 10mm biopsy punch. The samples were immediately fixed using 4%formaldehyde at 4° C. (overnight) followed by transfer to ethanol andembedding of the sample into a paraffin block. The blocks were thensectioned at 6 μm thickness by microtome (Leica) and underwentHematoxylin and Eosin (H&E) staining. For quantification of cellinfiltration within the hydrogels and immune response surrounding thehydrogels, a series of 3 random high power (40×) fields (HPFs) wereexamined for each section. Samples were analyzed for cell infiltration(>0.1 mm into the gel) by counting the total number of cells of any typewithin the injected hydrogels (N=5 with a sum of cells in 3 sectionsanalyzed per wound). Greater than 95% of the cells infiltrating the gelswere neutrophils. To measure immune response, the average of 3 HPFs fromdifferent sections of the wound were examined. The total number ofleukocytes/HPF within 0.2 mm of the hydrogel at the wound edge wasquantified and averaged for each wound type. The leukocyte count foreach wound was compared to its bilateral control on the same animal andthe relative difference was recorded as a fraction of each animal'soverall immune response. This comparison was possible because eachanimal had one wound injected with the microgel scaffold and one woundwith the non-porous control.

Wounds were imaged daily to follow closure of the wounds. Each woundsite was imaged using high-resolution camera (NIKON® COOLPIX®). Closurefraction was determined by comparing the pixel area of the wound to thepixel area within the 10 mm center hole of the red rubber splint.Closure fractions were normalized to Day 0 for each mouse/scaffold type(FIG. 7B).

At Day 5, mice (N=6) were sacrificed and tissue collected as in day 1mice. The samples were immediately submerged in TISSUE-TEK® OptimalCutting Temperature (OCT) fluid and frozen into a solid block withliquid nitrogen. The blocks were then cryo-sectioned at 25 μm thicknessby cryostat microtome (Leica) and kept frozen until use. The sectionswere then fixed with paraformaldehyde for 30 minutes at roomtemperature, hydrated with PBS, and kept at 4° C. until stained.

Slides containing tissue sections were either blocked with 3% normalgoat serum (NGS) in 1×PBS+0.05% Tween-20 (PBST) or simultaneouslyblocked and permeabilized with 0.2% TRITON® X-100 in 3% NGS in 1×PBSTfor sections stained with anti keratin-5 only. Sections were then washedin 3% NGS in 1×PBST. Primary antibody dilutions were prepared as followsin 3% NGS in 1×PBST:

rat anti mouse CD11b (BD Pharmingen)—1:100

rat anti mouse PECAM-1 (BD Pharmingen)—1:100

rabbit anti mouse NG2 (Millipore)—1:100

rabbit anti mouse keratin 5 (Covance, Inc.)—1:250

Sections were stained with primary antibodies overnight at 4° C., andsubsequently washed with 3% NGS in 1×PBST. Secondary antibodies were allprepared in 3% NGS in 1×PBST at a dilution of 1:100. Sections wereincubated in secondary antibodies for 1 hour at room temperature, andsubsequently washed with 1×PBST. Sections were counterstained with 2μg/ml DAPI in 1×PBST for 30 mins at room temperature. Sections weremounted in Antifade Gold mounting medium.

Confocal z-stack images acquired from day 5 tissue sections from bothnon-porous and microgel scaffold tissue blocks were compressed intoMIPs, followed by separation into individual images corresponding toeach laser channel (i.e., Gel, DAPI, CD11b). The gel channel image wasused to trace the edge of the gel-tissue interface using Adobeillustrator. The width of this line was expanded 75 μm both into thetissue and into the gel from the interface (150 μm in total thickness).The new edges of this line were then used to crop the original DAPI andCD11b images, to capture only the areas corresponding to +/−75 μm fromthe tissue gel interface. These images were then imported into ImageJ,and overlaid to merge the DAPI and CD11b channels into a single image.This image was analyzed using the cell counter plugin from ImageJ, whereboth the total number of nuclei was quantified, as well as the totalnumber of CD11b+ cells. Finally, the fraction of nuclei with acorresponding CD11b+ signal were reported for both within the tissue andwithin the gel.

FIG. 11 illustrates one example of method of treating damaged tissue102. FIG. 11 illustrates a wound site 100 formed in tissue 102 of amammal. In operation 500, a delivery device 110 (e.g., tube asillustrated) that contains therein the slurry of microgel particles 12contained in an aqueous solution is used to deliver the microgelparticles 12 to the wound site 100. Next, as seen in operation 510, anoptional applicator 122 is used to spread the microgel particles 12 intoand over the wound site 100. The applicator 122 is also used to make theupper, exposed surface of the microgel particles 12 generally flush withthe surface of the tissue 102. The applicator 122 can also be used tomake the upper, exposed surface of the microgel particles 12 mounded orelevated with respect to the surface of the tissue 102 to allow forincreased structure for cellular ingrowth and prevention of a depressedtissue interface upon full healing. Next, as seen in operation 520,annealing of the microgel particles 12 is initiated to form the scaffold10 of annealed microgel particles 12. In this particular example, alight source 124 in the form of a flashlight is used to illuminate amixture of microgel particles 12, a photoinitiator (e.g., Eosin Y), anda free radical transfer agent (e.g., RGD peptide). Of course, otherannealing modalities as described herein may also be used. The annealingreaction illustrated in FIG. 11 causes the formation of acovalently-stabilized scaffold 10 of microgel particles 12 havinginterstitial spaces therein. Cells 106 (as seen in FIG. 2C) from thesurrounding tissue 102 then begin to infiltrate the spaces within thescaffold 10, grow, stimulate, and ultimately effectuate the healingprocess of the tissue 102. In one embodiment, following the annealingreaction a bandage or moist dressing is optionally placed over thescaffold-filled wound to protect it from damage during the healingprocess. After a period of elapsed time, as illustrated in operation530, the scaffold 10 has degraded and the tissue 102 has returned to ahealed state.

In order to assess the ability of the porous gel scaffold to supportcell growth and network formation, an in vitro cell morphology andproliferation model was developed using three human cell lines: DermalFibroblasts (HDF), Adipose-derived Mesenchymal Stem Cells (AhMSC), andBone Marrow-derived Mesenchymal Stem Cells (BMhMSC). A single-cellsuspension was dynamically incorporated within a FXIIIa annealed porousgel scaffold. The three cell lines exhibited high cell viability (≧93%,FIG. 12B) following twenty-four (24) hours of culture within the porousgel scaffold.

Cells incorporated into the porous gel scaffold began to exhibit spreadmorphology ninety (90) minutes following the onset of annealing. Aftertwo (2) days in culture, all observed cells within the porous gelscaffolds exhibited a completely spread morphology, which continuedthrough day six. Importantly, an extensive network formation for allcell lines was observed by day two. Cell networks increased in size andcomplexity through the entirety of the experiment. The BMhMSCs were ofparticular note, as their expansive network formation and slowerproliferation rate indicated that these cells were able to spread toextreme lengths, forming highly interconnected cellular networks withinthe microporous scaffolds as seen in FIG. 12A.

The microgel particles 12 can be combined and mixed with a solution ofliving cells 106 prior to annealing to create a microporous scaffold 10that contains living cells 106 residing in the microporous network anddispersed either homogenously or heterogeneously within the macroscopicannealed gel scaffold 10 as seen in FIG. 13A.

The microgel particles 12 can be purified into an aqueous solution ofisotonic cell culture media for storage and when used to form a porousgel were annealed to one another via a non-canonical amide linkagebetween the K and Q peptides mediated by activated Factor XIII (FXIIIa),a naturally occurring enzyme responsible for stabilizing blood clots.This enzyme-mediated annealing process, allowed incorporation of livingcells 106 into a dynamically forming porous scaffold 10 that containedinterconnected microporous networks. Following addition of FXIIIa, butprior to scaffold annealing, a slurry of the microgel particles 12 canbe delivered via syringe application (FIG. 13A), ultimately solidifyingin the shape of the cavity in which they are injected as seen in FIGS.13B-E.

Microfluidic fabrication of the microgel particles 12 enablesdeterministic control over the microgel size and production frequency asillustrated in FIG. 14A. The pressure that is applied to the inlets ofthe microfluidic system 20, determines the frequency of microgelproduction (FIG. 14B). Further, porous microgel scaffolds 10 createdusing different size microgel particles 12 have distinct porouscharacteristics, such as the median pore size within the network as seenin FIG. 14C.

While embodiments have been shown and described, various modificationsmay be made without departing from the scope of the inventive conceptsdisclosed herein. The subject matter described herein, therefore, shouldnot be limited, except to the following claims, and their equivalents.

1-66. (canceled)
 67. A method of providing a scaffold for living tissuecomprising: delivering to the living tissue a flowable solutioncomprising a plurality of spherical microgel building blocks, whereinthe spherical microgel building blocks have a molecular mass between 481teradaltons and 60 petadaltons; exposing the spherical microgel buildingblocks to an annealing agent that crosslinks the spherical microgelbuilding blocks to adjacent spherical microgel building blocks andadjacent living tissue at points of contact to form acovalently-stabilized scaffold with a molecular mass greater than 60petadaltons and having interstitial spaces throughout; and promotingcells from the living tissue to migrate into the interstitial spaces ofthe covalently-stabilized scaffold.
 68. The method of claim 67, whereinthe flowable solution of spherical microgel building blocks is deliveredto the living tissue by injection from a syringe.
 69. The method ofclaim 67, wherein the plurality of spherical microgel building blockshave a distribution of diameters with a coefficient of variation lessthan 50%.
 70. The method of claim 67, wherein the volume fraction of theswollen spherical microgel building blocks before crosslinking isbetween 30% and 99%.
 71. The method of claim 67, wherein the sphericalmicrogel building blocks comprise a polyethylene glycol component andpeptide component.
 72. The method of claim 71, wherein the plurality ofspherical microgel building blocks comprise a first plurality ofspherical microgel building blocks comprising L amino acids and a secondplurality of spherical microgel building blocks comprising D aminoacids.
 73. The method of claim 71, wherein the peptide componentcomprises an MMP-degradable peptide.
 74. The method of claim 72, whereinthe ratio between the first plurality of spherical microgel buildingblocks and the second plurality of spherical microgel building blocks issubstantially 1:1.
 75. The method of claim 74, wherein a portion of thespherical microgel building blocks remain in the living tissue after 21days.
 76. The method of claim 75, wherein the living tissue comprises anepidermal wound and wherein the covalently-stabilized scaffold increasesthe formation of sweat glands and hair follicles in thecovalently-stabilized scaffold after 21 days.
 77. The method of claim75, wherein the living tissue comprises an epidermal wound and whereinthe migrated cells increase dermal thickness in thecovalently-stabilized scaffold after 21 days.
 78. The method of claim77, wherein the dermal thickness increases at least 2-fold.
 79. Themethod of claim 67, wherein exposing the spherical microgel buildingblocks to an annealing agent comprises exposing the spherical microgelbuilding blocks to light, and wherein the flowable solution of sphericalmicrogel building blocks further comprises Eosin Y.
 80. A method ofproviding a scaffold for living tissue comprising: delivering to theliving tissue a flowable solution of polymer building blocks to form apack of polymer building blocks in physical contact with each other andadjacent living tissue, wherein the polymer building blocks have amolecular mass between 481 teradaltons and 60 petadaltons, wherein thepolymer building blocks comprise a polyethylene glycol component andpeptide component; exposing the polymer building blocks to light thatcrosslinks the polymer building blocks to adjacent polymer buildingblocks and adjacent living tissue at points of physical contact to forma covalently-stabilized scaffold having interstitial spaces therein; andpromoting cells from the living tissue to migrate into the interstitialspaces of the covalently-stabilized scaffold.
 81. The method of claim80, wherein the interstitial spaces comprise an interconnected networkof continuously connected voids.
 82. The method of claim 81, wherein thevolume of the interconnected network of voids is between 10% and 50% ofthe entire volume of the covalently-stabilized scaffold.
 83. The methodof claim 81, wherein the characteristic length scale of each void isbetween 12 μm and 27 μm.
 84. The method of claim 81, wherein the voidshave walls exhibiting negative concavity.
 85. The method of claim 80,wherein the polymer building blocks comprise a first type of polymerbuilding block with a first peptide component and a second type ofpolymer building block with a second peptide component.
 86. The methodof claim 85, wherein the ratio of the first type of polymer buildingblocks and the second type of polymer building blocks is substantiallyequal.
 87. The method of claim 85, wherein the first peptide componentcomprises at least one D amino acid and wherein the second peptidecomponent comprises L amino acids.